{"title":"Aurora Biolabs","description":"Products supplied by Aurora Biolabs.","products":[{"product_id":"tr-fret-cereblon-4c-binding-assay-kit-bht20700004","title":"TR-FRET Cereblon-4C Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eE3 ubiquitin ligases are a large family of enzymes that catalyze the critical final step in the ubiquitination \ncascade. Cereblon (CRBN) serves as the substrate-recognition component of the E3 ubiquitin ligase \n\ncomplex CRBN\/DDB1\/CUL4A\/RBX1 (CRBN-4C). CRBN is one of the most widely utilized E3 ligases \nin the design of PROTACs (Proteolysis-Targeting Chimeras) for targeted protein degradation (TPD) in \n\ndrug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe TR-FRET CRBN Binding Assay Kit is designed to measure the binding affinity between CRBN and \nits ligands. The kit includes Tag1-CRBN-4C (a complex of CRBN\/CUL4A\/DDB1\/RBX1), a terbium-\nlabeled anti-Tag1 antibody, and a fluorescently labeled CRBN ligand, thalidomide. When CRBN binds \nto thalidomide, the terbium donor (on the anti-Tag1 antibody) is brought into close proximity to the \nfluorescent acceptor (fluorophore-labeled thalidomide), resulting in fluorescence resonance energy \ntransfer (FRET). The binding interaction can be quantitatively measured as an HTRF signal, calculated \nfrom the ratio of the emission intensities at 665 nm (acceptor) and 620 nm (donor). If a test compound \ncompetes with thalidomide for CRBN binding, the HTRF signal will decrease, indicating inhibition of \nCRBN–thalidomide interaction.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that bind to Cereblon for drug discovery. \n\n-4510 or 858453-5700 Fax: 855-898-3979 \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e272625-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-4C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eFluorescence-labeled Thalidomide\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e20 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare CRBN-4C solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare FL-Thalidomide solution\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 20 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate sample HTRF signal of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare CRBN-4C solution Thaw CRBN-4C protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: CRBN-4C protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the CRBN-4C protein 20-fold (1µL CRBN-4C + 19 µL assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of assay buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of assay buffer to each of negative and positive control wells. If the compound is diluted in 10% DMSO, add 2 µl of assay buffer containing 10% DMSO to each of\tnegative and positive control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare FL-Thalidomide solution Dilute FL-Thalidomide 125-fold (1 µL FL-Thalidomide + 124 µL of assay buffer). Add 4 µl of diluted FL-Thalidomide solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag1 antibody 1:200 in assay buffer. For example: 1 µl of Terbium- labeled anti-Tag1 antibody + 199 µl of assay buffer. Add 10 µl of this dye mixture to each well. -4510 or 858453-5700 Fax: 855-898-3979 3\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Incubate the reaction at room temperature for 20 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eUbiquitin-Proteasome \/ Targeted Protein Degradation\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology; Hematology; PROTAC Drug Discovery\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238301983085,"sku":"272625","price":1299.0,"currency_code":"USD","in_stock":false}]},{"product_id":"sars-cov-2-nucleocapsid-protein-binding-kit-for-rabbit-antibody-bht20700029","title":"SARS-CoV-2 Nucleocapsid Protein Binding Kit (For rabbit antibody)","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eSARS-CoV-2 Nucleocapsid protein (NP) is one of the core components of SARS-CoV-2 virus. It forms \na complex with viral genomic RNA in a helical symmetrical structure and plays a key role in the process \nof virus replication and assembly. Since NP is abundantly expressed during infection, it can be used \nas an important diagnostic marker for COVID-19 and also can be used as a potential drug target or \ndeveloping vaccines.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe SARS-CoV-2 Nucleocapsid protein (NP) Binding kit is a TR-FRET based assay, that is designed \nto detect binding status of NP to the test antibody. Terbium-labeled anti-Tag1 antibody serves as \nfluorescence donor, that binds to the His-Tagged NP. If a test rabbit antibody binds to NP, fluorescence-\nlabeled anti-rabbit antibody (fluorescence acceptor) will be brought in close proximity with the \nfluorescence donor. Excitation of Terbium (340 nm) generates fluorescence resonance energy transfer \n(FRET) to the fluorescence-labeled acceptor, which consequently fluoresces at 665 nm (figure below). \nThus, the test antibody binding to NP can be quantitively measured by calculation of the fluorescent \nratio of 665 nm\/620 nm.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of antibodies that bind to NP.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eAurora Biolabs LLC, San Diego, CA 92121, USA; www.aurorabiolabs.com; SARS-CoV-2 Nucleocapsid Protein Binding Kit (for rabbit antibody) A HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e2x Assay Buffer\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003eCustomer Test anti-NP-rabbit antibody (to be tested antibody)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare SARS-CoV-2 Nucleocapsid protein Dilute SARS-CoV-2 Nucleocapsid protein (NP) 1,500-fold with 1X DTT-containing assay buffer. For example: 1 µl of NP + 1,499 µl of 1X DTT-containing assay buffer. Add 5 µl of diluted NP protein to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare Antibody solution\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag1 Ab and fluorescence-labeled anti-rabbit antibody 1:200 in 1X DTT- containing assay buffer. For example: 1 µl of Terbium-labeled anti-Tag1 Ab + 1 µl of fluorescence- labeled anti-rabbit antibody + 198 µl of 1X DTT-containing assay buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Incubate the reaction at room temperature for 1 hour.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample signal. Calculate percentage activity \n\nAurora Biolabs LLC, San Diego, CA 92121, USA; www. SARS-CoV-2 Nucleocapsid Protein Binding Kit \n(for rabbit antibody) In the absence of the compound (positive control), the sample signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% activity. The \npercent activity in the presence of each compound is calculated according to the following \nequation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence of the \ncompound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e Nucleocapsid Protein Binding\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302015853,"sku":"728273","price":1799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"eif4e-eif4g-binding-assay-kit-bht20700005","title":"eIF4E\/eIF4G Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eEukaryotic translation initiation factor eIF4E, the mRNA cap-binding protein, is considered the rate-\nlimiting factor in translation. It plays an important role in cap-dependent translation initiation and \nrecruitment of mRNA to ribosomes. Overexpression of eIF4E has been documented in numerous \nhuman cancers and contributes to transformation, tumorigenesis, and progression of cancers. \nTherefore, eIF4E is an attractive drug target for cancer treatment.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe eIF4E binding assay kit, a TR-FRET based assay, is designed to screen compounds that bind to \neIF4E. A fluorescence-labelled tracer and the N-terminal tagged full-length human eIF4E\/eIF4G \ncomplex are used in this assay kit. A Terbium-labeled antibody binding to the tag on eIF4E serves as \na fluorescence donor (HTRF donor). The binding of the fluorescence-labeled tracer to the eIF4E brings \nTerbium on the anti-Tag antibody close to the fluorophore on the tracer (HTRF acceptor). Activation of \nthe Terbium results in fluorescence resonance energy transfer (FRET). Thus, the binding status can \nbe quantitively measured by calculating the ratio of the emission fluorescence intensity of the acceptor \n(665 nm) and donor (620 nm). The competitive binding of a non-fluorescence compound will reduce \nthe FRET signal.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that bind to eIF4E\/eIF4G.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eAurora Biolabs LLC, San Diego, CA 92121, USA; www.aurorabiolabs.com; 1 A HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e34343-BK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e16 µL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare assay buffer containing 1 mM DTT\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare eIF4E\/eIF4G solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare fluorescence-labeled tracer\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 60 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate the ratio of the fluorescent intensity of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare eIF4E\/eIF4G solution Thaw eIF4E\/eIF4G protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: eIF4E\/eIF4G protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the eIF4E\/eIF4G protein 200-fold (1 µL eIF4E\/eIF4G + 199 µL 1X assay buffer containing DTT). Add 8 µl of diluted protein solution to each positive control wells and inhibitor test wells. Add 8 µl of 1X DTT containing buffer to each of negative control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each negative and positive control wells. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare fluorescence-labeled tracer Thaw the tracer at room temperature. Dilute the tracer 125-fold (1 µL of 1 M tracer + 124 µL 1X assay buffer containing DTT). Add 5 µl of diluted tracer to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag antibody 1:200 (1 µl of Terbium-labeled anti-Tag antibody + 199 µl of 1X DTT-containing assay buffer). Add 5 µl of this dye mixture to each well. Dilute just enough of the antibody for each reaction set. Store remaining undiluted antibody at -80°C. Do not re-use the diluted antibody.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 60 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eCap-Dependent Translation Initiation\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology (eIF4E-overexpressing tumors)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302048621,"sku":"34343-BK","price":2599.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-wt-nucleotide-exchange-assay-kit-bht20700009","title":"Kras WT Nucleotide Exchange Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \n\nsignaling pathways. The Ras signaling pathway plays an important role in cell proliferation and \n\ndifferentiation. Conversion of Kras from the inactive GDP-bound state to the active GTP-bound state \n\ntriggers the downstream effector and promotes cell growth. RAS genes are frequently mutated in \n\nvarious human tumors. These mutations block the GTPase activity of RAS and lock RAS in the GTP-\n\nbound state, resulting in constitutively active signals through the downstream cascades leading to \n\ncancer cell proliferation.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (wild type, WT) nucleotide exchange assay is a TR-FRET based assay. The assay kit is \n\ndesigned to detect the GTP binding status of wild type Kras in the presence of SOS1, the most-studied \n\nguanine nucleotide exchange factor (GEF) of Kras. The Tag2 Kras in this assay kit is recognized by a \n\nTerbium-labeled anti-Tag2 antibody (HTRF donor). If Kras binds to a fluorescence-labeled GTP (HTRF \n\nacceptor), the donor and the acceptor will be brought in close proximity. Excitation of Terbium (340 nm) \n\ngenerates fluorescence resonance energy transfer (FRET) to the fluorescence-labeled GTP acceptor, \n\nwhich consequently fluoresces at 665 nm (figure below). Thus, GTP binding to Kras can be quantitively \n\nmeasured by calculation of the fluorescent ratio of 665 nm\/620 nm. \n\nAurora Biolabs LLC, San Diego, CA 92121; www.aurorabiolabs.com; \n\nKras (WT) Nucleotide Exchange Assay Kit\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit Kras activation for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-NK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e20 µL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003e1. Prepare 1X assay buffer containing 1 mM DTT (1X DTT-containing assay buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare SOS1 solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare Kras solution\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 20 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare 1X assay buffer containing 1 mM DTT (1X DTT-containing assay buffer) For example, mix 996 µl distilled water with 1000 µl of 2X assay Buffer (Catalogue number: 5727- NK-B) and 4 µl of 0.5 M DTT. Make only enough 1X DTT-containing assay buffer as needed for the assay. Store the remaining 2X assay buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare SOS1 solution Thaw SOS1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: SOS1 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the SOS1 protein 400-fold (1 µL SOS1 + 399 µL 1X DTT-containing assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of 1X DTT-containing assay buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare Kras solution\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:200 and dilute fluorescence-labeled GTP 1:40 in 1X DTT-containing assay buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 5 µl of fluorescence-labeled GTP + 194 µl of 1X DTT-containing assay buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 20 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e17 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302081389,"sku":"5727-4121NK","price":1699.0,"currency_code":"USD","in_stock":false}]},{"product_id":"pd-1-pd-l1-inhibitor-binding-assay-kit-bht20700003","title":"PD-1\/PD-L1 Inhibitor Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eImmune checkpoint blockade is a groundbreaking approach in cancer immunotherapy that enhances \n\nthe immune system’s ability to recognize and destroy cancer cells. The immune checkpoint PD-\n\n1(CD279)\/PD-L1 (CD274 or B7-H1) is an attractive target for cancer immunotherapy. The interaction \n\nof PD-1 with PD-L1 induces T cell apoptosis and allows cancer to evade the immune response by \n\nsuppressing the adaptive immune system. Immunotherapy drugs work by blocking the interaction \n\nbetween PD-1 and PD-L1, and enhancing the anti-tumor immune response.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe PD-1\/PD-L1 binding assay kit is a TR-FRET based assay, which is designed to detect the binding \n\nstatus between PD-1 and PD-L1. Tag6-PD-1 and Tag7-PD-L1 are included in this assay kit. Binding of \n\nTag6-PD-1 to Tag7-PD-L1 brings the Terbium (Tb, HTRF donor) and the fluorophore d2 (HTRF \n\nacceptor) in a proximity distance, and activation of Tb results in fluorescence resonance energy transfer \n\n(FRET). Thus, the binding status can be quantitively measured by calculating the ratio of the emission \n\nfluorescence intensity of the acceptor (665 nm) and donor (620 nm). Interference of the PD-1\/PD-L1 \n\nbinding will reduce the HTRF signal.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003ePD-1\/PD-L1 Inhibitor Binding Assay Kit \n High throughput screening of compounds that inhibit the binding between PD-1 and PD-L1 for drug \ndiscovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e234822-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare PD-1 solution Thaw PD-1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: PD-1 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the PD-1 protein 32-fold (1 µL PD-1 + 31 µL 1X assay buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare PD-L1 solution Thaw PD-L1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: PD-L1 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the PD-L1 protein 400-fold (1 µL PD-L1 + 399 µL 1X assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of assay buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag6 antibody and fluorescence-labeled anti-Tag7 antibody 1:200 in assay buffer. For example: 1 µl of Terbium-labeled anti-Tag6 antibody + 2 µl of fluorescence- labeled anti-Tag7 antibody + 197 µl of assay buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 1 hour.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample HTRF signal (P) is defined as \n100% activity. In the absence of enzyme (negative control), the sample HTRF signal (N) is\n defined as 0% activity. The percent activity in the presence of each compound is calculated \naccording to the following equation: % activity = (S-N)\/(P-N) X100, where S= the sample HTRF \nsignal in the presence of the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e0.47 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eReference Compound:\u003c\/em\u003e Pembrolizumab\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eImmune Checkpoint (T cell exhaustion pathway)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology \/ Immunotherapy\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302146925,"sku":"237352","price":1699.0,"currency_code":"USD","in_stock":false}]},{"product_id":"dna-polymerase-theta-activity-assay-kit-bht20700006","title":"DNA Polymerase Theta Activity Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eDNA polymerase theta (Pol θ) is involved in an end-joining pathway of DNA double-strand breaks. Over \nexpression of Pol θ is found in many cancers, including stomach, colon, breast and lung cancers, and \ncorrelated with poorer patient survival. Because suppression of gene expression of Pol θ results in \nsensitivity of cells to ionizing radiation and some DSB-inducing drugs, Pol θ is a validated anti-cancer drug \ntarget. \n\nDescription \n\nThe Aurora DNA Polymerase Theta activity assay kit is a homogeneous fluorescence-based assay for \nscreening inhibitors that block DNA polymerase activity of DNA Pol θ. \n\n The assay is fast, convenient, and requires just two steps. In the first step, the DNA Pol θ enzyme \nsynthesizes double-stranded DNA using a DNA template in the presence of dNTP. In the second step, a dye \nthat binds to double-stranded DNA is added to the solution resulting in the increase of fluorescence, \nintensity of which can be measured with a fluorescent plate reader at the excitation wavelengths of 495 \nnm and emission wavelengths of 525 nm. \n\nMaterials supplied \n\nCatalogue Number \n362201 \n362204 \n4687 \n362003 \n4930 \n362202 \n\nItem \n2X Assay Buffer \n20 µM DNA template \n10 mM dNTP \nRecombinant DNA Pol θ CTD \nDye solution \nStop solution \nBlack low binding 96 well plate \n\nAmount \n25 mL \n7 µL \n5 µL \n5 µL \n15 µL \n3 mL \n1 \n\nStorage \n-20°C \n-20°C \n-20°C \n-80°C \n-20°C \n-20°C \nRT \n\nMaterials Needed but not supplied \n\nA microplate reader capable of measuring fluorescence at excitation wavelengths of 495 nm and \nemission wavelengths of 525 nm. \n\n1. 0.5 M DTT \n2. Adjustable micro-pipettor \n3. Sterile Tips \n\nStability \n\n12 months if stored under the indicated conditions. \n\nAurora Biolabs LLC, San Diego, CA 92121, USA. www.aurorabiolabs.com, \n\n \n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare 1X buffer containing 1 mM DTT.\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution.\u003c\/li\u003e\n\u003cli\u003ePrepare DNA Pol Theta solution.\u003c\/li\u003e\n\u003cli\u003eAdd the inhibitor solution\u003c\/li\u003e\n\u003cli\u003eIncubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli\u003ePrepare substrate solution\u003c\/li\u003e\n\u003cli\u003eIncubate the plate at 30°C for 1 hour.\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure the fluorescent intensity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution. If the inhibitor compound is dissolved in water, make a solution of the compound 5-fold higher than the final concentration in 1X assay buffer (since you will add 5 µl to the 25 µl reaction). Then make a series of dilutions in 1X assay buffer from this solution to your preferred concentrations. If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 20-fold dilution in 1X assay buffer (at this step, the compound concentration is 5-fold higher than the final concentration and the DMSO concentration is 5%). Then make a series of dilutions in 5% of DMSO from this solution to your preferred concentrations. Since 5 µl of the compound solution will be added to the 25 µl reaction, the final concentration of DMSO in all of reactions is 1%.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare DNA Pol Theta solution. Thaw DNA Pol θ CTD enzyme (catalogue number 362003) on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: DNA Pol θ CTD enzyme is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted enzyme. Dilute DNA Pol Theta enzyme 650-fold (1:650) in 1X assay buffer with 1 mM DTT. Add 10 µl of diluted enzyme solution to each of positive control well and inhibitor test well. Add 1X buffer to each of background well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add the inhibitor solution Add 5 µl of 1X assay buffer to each background well and positive control well if the inhibitor is diluted in 1X buffer. Add 5 µl of 1X assay buffer with 5% DMSO to each of background well and positive control well if the inhibitor is diluted in 1X buffer with 10% DMSO. Add 5 µl of diluted inhibitor solution from Step 2 to each of the inhibitor test well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare substrate solution During the incubation of the enzyme and the inhibitor solution, prepare substrate solution containing 0.125 µM DNA template (dilute from 20 µM DNA template, catalogue number 362004) and 25 µM dNTP (dilute from 10 mM dNTP) in 1X assay buffer. Make only enough solution as need for the assay. Store the remaining 20 µM DNA template and 10 mM dNTP solution to -20°C. Add 10 µl of the substrate solution to each of well, including background wells, positive control wells and the inhibitor test wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the plate at 30°C for 1 hour.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Prepare dye solution Dilute the Dye solution 200-fold in Stop solution (catalogue number 362202). Make only enough solution as need for the assay. Store the remaining Dye solution to -20°C. Add 25 µl the Dye solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Incubate at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Measure the fluorescent intensity Measure the fluorescent intensity at the excitation wavelengths of 495 nm and the emission wavelengths of 525 nm. Positive Control Inhibitor Test\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate percentage activity of the enzyme \n\n% Activity= \n\n(Fp – Fb) – (Fi-Fb) \nFp - Fb \n\nX 100 \n\nWhere Fp refers to fluorescent intensity of the positive control, Fb refers to fluorescent intensity of \nbackground, and Fi refers to fluorescent intensity of the inhibitor. Graph the percentage activity as a function of the inhibitor concentration to determine the IC50 of the test \ninhibitor. No CPD refers to no compound control \n(compound vehicle only).\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e19 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eDNA Double-Strand Break Repair (End-Joining TMEJ)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology (POLQ-overexpressing cancers: breast; lung; colon; stomach)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"96 reactions","offer_id":53238302114157,"sku":"362101-96","price":839.0,"currency_code":"USD","in_stock":false},{"title":"384 reactions","offer_id":53238305554797,"sku":"362101-384","price":1199.0,"currency_code":"USD","in_stock":false}]},{"product_id":"tr-fret-parp1-trapping-assay-kit-bht20700024","title":"TR-FRET PARP1 Trapping Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eTR-FRET PARP1 Trapping Assay Kit \n PARP1 (Poly (ADP-ribose) polymerase 1) is an abundant member of the PARP family and plays a \ncrucial role in DNA repair by acting as a damage sensor and facilitator. It binds to DNA at the site of \ndamage, becomes catalytically activated, and uses NAD⁺ as a substrate to add poly (ADP-ribose) \n(PAR) chains to itself and other proteins—a process called PARylation that results in the recruitment \nof other DNA repair proteins to the damaged site. Because of the high negative charge of PAR \npolymers, extensive autoPARylation of PARP1 leads to the dissociation of PARP1 from DNA, which is \nrequired for DNA repair completion. PARP1 is often overexpressed in various cancers, including breast, \novarian, prostate, lung, and glioblastoma. This overexpression is thought to support tumor cell survival. \nSome PARP inhibitors not only block the catalytic activity of PARP1 but also trap PARP1 on DNA at \nsites of damage, preventing its release. This creates a toxic DNA-protein complex that interferes with \nDNA replication and repair, leading to cell death—particularly in cancer cells deficient in homologous \nrecombination repair (e.g., BRCA1\/2-mutant cells).\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe TR-FRET PARP1 Trapping Assay Kit is designed to detect the poly-ADP-ribosylation activity of \nPARP1 and the status of PARP1 trapping on DNA. The DNA substrate in the kit is labeled with a \nfluorophore (acceptor). A Terbium (Tb)-labeled anti-Tag2 antibody binding to Tag2-Kras serves as \nthe fluorescence donor. Activation of Tb results in fluorescence resonance energy transfer (FRET) if \nPARP1 binds to the fluorescence-labeled DNA, since the binding brings the fluorescence donor into \nclose proximity with the fluorophore acceptor. Thus, the binding status can be quantitatively \nmeasured by calculating the ratio of the emission fluorescence intensities of the acceptor (665 nm) \nand the donor (620 nm). In the presence of NAD⁺, auto-PARylation of PARP1 leads to dissociation of \nPARP1 from the DNA, resulting in a decrease in the FRET signal. Conversely, inhibition of auto-\nPARylation activity traps PARP1 on the DNA, and the FRET signal remains high.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eTR-FRET PARP1 Trapping Assay Kit \n High throughput screening of compounds that inhibit the auto-PARylation activity of PARP1 for drug \ndiscovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e7277-TAK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare PARP1 solution Thaw PARP1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: PARP1 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the PARP1 protein 500-fold (1 µL PARP1 + 499 µL assay buffer). Add 4 µl of diluted protein solution to each of positive control wells and inhibitor test wells. Add 4 µl of assay buffer to each of negative control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare the DNA substrate solution Dilute the fluorescence-labeled DNA 100-fold (1 µL DNA + 99 µL assay buffer). Add 4 µl of the diluted DNA solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare NAD+ solution Dilute the NAD+ 50-fold (1 µL NAD+ + 49 µL assay buffer). Add 5 µl of diluted NAD+ solution to each of positive control and compound test wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:100. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 99 µl assay buffer. Add 5 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e TR-FRET PARP1 Trapping Assay\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e1.9 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eReference Compound:\u003c\/em\u003e Talazoparib\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eDNA Damage Response (DDR); PARP-mediated repair\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology (breast; ovarian; prostate; BRCA-mutant tumors)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302212461,"sku":"72771TAK","price":1799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12d-craf-cypa-inhibitor-assay-kit-bht20700014","title":"Kras G12D\/cRAF\/CYPA\/Inhibitor Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \nhuman tumors, the Ras-RAF signaling pathway is considered an important therapeutic target for cancer \n\ntreatment. However, Ras is considered undruggable since it lacks suitable binding pockets on the \nsurface. Recently, a discovery of a small molecule inhibitor blocks Ras-RAF signaling pathway by \n\nremolding Cyclophilin A (CYPA) and forming a CYPA:drug:KRAS ternary complex. This inhibitory \nstrategy provides a new method for developing drugs targeting Kras for treatment of cancers.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12D) Inhibitor assay kit is a TR-FRET based assay, which is designed to screen Kras \n\ninhibitors and determine the Kras-inhibitor binding affinity. Tag2-Kras (G12D) in this assay kit is loaded \nwith GppNHp, which represents the activated Kras. The Ras binding domain (RBD) of cRAF has a \nTag1 at N-terminus. A Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a \nfluorescence donor (HTRF donor), activation of which results in fluorescence resonance energy \n\ntransfer (FRET) if Tag1-cRAF binds to the Kras, since the binding brings Terbium on the anti-Tag2 \nantibody close to the fluorophore on the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status \ncan be quantitively measured by calculating the ratio of the emission fluorescence intensity of the \nacceptor (665 nm) and donor (620 nm). If an inhibitor associated with CYPA binds to the Kras and \nblocks the cRAF binding, the HTRF signal will be reduced. \n\n-4510 or 858453-5700 Fax: 855-898-3979 \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (G12D) and \ncRAF for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-CK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000, Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare compound dilution buffer containing 2 mM DTT (CD buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare 1X Assay Buffer containing 2 mM DTT (AB buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare Kras (G12D) solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare cRAF solution\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate sample HTRF signal of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in CD buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in CD buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in CD buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare 1X Assay Buffer containing 2 mM DTT (AB buffer) For example, mix 500 µl of 2X Kras Binding Buffer, 496 µl of distilled water and 4 µl of 0.5 M DTT. Make only enough AB buffer as needed for the assay. Store the remaining Binding buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare Kras (G12D) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein 75-fold (1µL Kras G12D + 74 µL AB buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of AB buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of CD buffer to each of negative and positive control well. -4510 or 858453-5700 Fax: 855-898-3979 3\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare cRAF solution Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 400-fold (1 µL cRAF + 399 µL of AB buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody and fluorescence-labeled anti-Tag1 antibody 1:200 in AB buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 1 µl of fluorescence-labeled anti-Tag1 antibody + 198 µl of AB buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e2.4 μM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eReference Compound:\u003c\/em\u003e RMC-9805\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302179693,"sku":"5727-4123CK","price":1999.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12c-craf-cypa-inhibitor-assay-kit-bht20700011","title":"Kras G12C\/cRAF\/CYPA\/Inhibitor Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \nhuman tumors, the Ras-RAF signaling pathway is considered an important therapeutic target for cancer \n\ntreatment. However, Ras is considered undruggable since it lacks suitable binding pockets on the \nsurface. Recently, a discovery of a small molecule inhibitor blocks Ras-RAF signaling pathway by \n\nremolding Cyclophilin A (CYPA) and forming a CYPA:drug:KRAS ternary complex. This inhibitory \nstrategy provides a new method for developing drugs targeting Kras for treatment of cancers.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12C) Inhibitor assay kit is a TR-FRET based assay, which is designed to screen Kras \n\ninhibitors and determine the Kras-inhibitor binding affinity. Tag2-Kras (G12C) in this assay kit is loaded \nwith GppNHp, which represents the activated Kras. The Ras binding domain (RBD) of cRAF has a \nTag1 at N-terminus. A Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a \nfluorescence donor (HTRF donor), activation of which results in fluorescence resonance energy \n\ntransfer (FRET) if Tag1-cRAF binds to the Kras, since the binding brings Terbium on the anti-Tag2 \nantibody close to the fluorophore on the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status \ncan be quantitively measured by calculating the ratio of the emission fluorescence intensity of the \nacceptor (665 nm) and donor (620 nm). If an inhibitor associated with CYPA binds to the Kras and \nblocks the cRAF binding, the HTRF signal will be reduced. \n\n-4510 or 858453-5700 Fax: 855-898-3979 \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (G12C) and \ncRAF for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-CK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000, Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare compound dilution buffer containing 2 mM DTT (CD buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare 1X Assay Buffer containing 2 mM DTT (AB buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare Kras (G12C) solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare cRAF solution\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate sample HTRF signal of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in CD buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in CD buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in CD buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare 1X Assay Buffer containing 2 mM DTT (AB buffer) For example, mix 500 µl of 2X Kras Binding Buffer, 496 µl of distilled water and 4 µl of 0.5 M DTT. Make only enough AB buffer as needed for the assay. Store the remaining Binding buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare Kras (G12C) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein 110-fold (1µL Kras G12C + 109 µL AB buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of AB buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of CD buffer to each of negative and positive control well. -4510 or 858453-5700 Fax: 855-898-3979 3\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare cRAF solution Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 400-fold (1 µL cRAF + 399 µL of AB buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody and fluorescence-labeled anti-Tag1 antibody 1:200 in AB buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 1 µl of fluorescence-labeled anti-Tag1 antibody + 198 µl of AB buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e0.26 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eReference Compound:\u003c\/em\u003e RMC-6291\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302310765,"sku":"5727-4122CK","price":1999.0,"currency_code":"USD","in_stock":false}]},{"product_id":"ido1-activity-assay-kit-for-inhibitor-screening-bht20700034","title":"IDO1 Activity Assay Kit for Inhibitor Screening","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eTryptophan is an essential amino acid that is involved in protein synthesis and regulation of the local \nimmune response by T lymphocytes. Indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes oxidation of \ntryptophan to N-formylkynurenine (NFK), the initial and rate limiting step in the pathway of catabolism \nof tryptophan. Over expression of IDO1 in variety of cancers results in the depletion of Tryptophan and \nthe accumulation of kynurenine, that have been proposed as mechanisms that contribute to the \nsuppression of the immune response.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eIDO1 Activity Assay Kit is designed to measure the activity of IDO1 enzyme and can be used for \nscreening IDO1 inhibitors. The activity assay is carried out on a 96-well plate. After incubation of the \nenzyme, substrate and inhibitors, absorbance of the product NFK is measure at 321nm, and IDO1 \nactivity is calculated based on the absorbance value. The kit contains enough solutions for 100 \nreactions.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of IDO1 inhibitors.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eSpectrophotometer capable of measuring absorbance at 321 nm.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eCatalog number\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare a series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare IDO1 protein solution Thaw IDO1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: IDO1 protein is sensitive to freeze\/thaw cycles. Limit the number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the IDO1 protein 50-fold (for example: 10 µL IDO1 + 490 µL 1X DTT-containing assay buffer). Add 80 µl of diluted protein solution to each of positive control well and inhibitor test wells. Add 80 µl of 1X DTT containing buffer to each of the negative control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor solution Add 20 µl of diluted inhibitor solution to each inhibitor test wells. Add 20 µl of 1X DTT-containing assay buffer to each positive and negative control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare Substrate solution Dilute Tryptophan solution 50-fold (for example: 10 µL Tryptophan + 490 µL 1X DTT-containing assay buffer). Add 100 µl of diluted substrate solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Incubate the reaction at 30°C for 2 hours.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Measure absorbance\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% activity. The \npercent activity in the presence of each compound is calculated according to the following \nequation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence of the \ncompound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e62 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eReference Compound:\u003c\/em\u003e INCB 024360\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eTryptophan Catabolism; Immune Suppression Pathway\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology \/ Immunotherapy\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"96 reactions","offer_id":53238302245229,"sku":"910010","price":799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12v-craf-cypa-inhibitor-assay-kit-bht20700019","title":"Kras G12V\/cRAF\/CYPA\/Inhibitor Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \nhuman tumors, the Ras-RAF signaling pathway is considered an important therapeutic target for cancer \n\ntreatment. However, Ras is considered undruggable since it lacks suitable binding pockets on the \nsurface. Recently, a discovery of a small molecule inhibitor blocks Ras-RAF signaling pathway by \n\nremolding Cyclophilin A (CYPA) and forming a CYPA:drug:KRAS ternary complex. This inhibitory \nstrategy provides a new method for developing drugs targeting Kras for treatment of cancers.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eKras (G12V)\/cRAF\/CYPA\/Inhibitor Assay Kit is a TR-FRET based assay, which is designed to screen \n\nKras inhibitors and determine the Kras-inhibitor binding affinity. Tag2-Kras (G12V) in this assay kit is \nloaded with GppNHp, which represents the activated Kras. The Ras binding domain (RBD) of cRAF in \nthe kit has a Tag1 at N-terminus. A Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves \nas a fluorescence donor (HTRF donor), activation of which results in fluorescence resonance energy \n\ntransfer (FRET) if Tag1-cRAF binds to the Kras, since the binding brings Terbium on the anti-Tag2 \nantibody close to the fluorophore on the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status \ncan be quantitively measured by calculating the ratio of the emission fluorescence intensity of the \nacceptor (665 nm) and donor (620 nm). If an inhibitor associated with CYPA binds to the Kras and \nblocks the cRAF binding, the HTRF signal will be reduced. \n\n-4510 or 858453-5700 Fax: 855-898-3979 \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (G12V) and \ncRAF for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-CK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000, Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare compound dilution buffer containing 2 mM DTT (CD buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare 1X Assay Buffer containing 2 mM DTT (AB buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare Kras (G12V) solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare cRAF solution\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate sample HTRF signal of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in CD buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in CD buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in CD buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare 1X Assay Buffer containing 2 mM DTT (AB buffer) For example, mix 500 µl of 2X Kras Binding Buffer, 496 µl of distilled water and 4 µl of 0.5 M DTT. Make only enough AB buffer as needed for the assay. Store the remaining Binding buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare Kras (G12V) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein 105-fold (1µL Kras G12V + 104 µL AB buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of AB buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of CD buffer to each of negative and positive control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare cRAF solution -4510 or 858453-5700 Fax: 855-898-3979 3 Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 400-fold (1 µL cRAF + 399 µL of AB buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody and fluorescence-labeled anti-Tag1 antibody 1:200 in AB buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 1 µl of fluorescence-labeled anti-Tag1 antibody + 198 µl of AB buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e98 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eReference Compound:\u003c\/em\u003e RMC-6236\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302343533,"sku":"5727-4128CK","price":1999.0,"currency_code":"USD","in_stock":false}]},{"product_id":"tev-protease-activity-assay-kit-bht20700001","title":"TEV Protease Activity Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eTobacco Etch Virus protease (TEV protease) is a highly sequence specific cysteine protease. It has a \nstrict 7 amino acid cleavage recognition sequence of Glu-Asn-Leu-Tyr-Phe-Gln ↓ (Gly\/Ser). The high \nspecificity makes this protease excellent for the removal of affinity-tags from purified recombinant \nproteins.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe TEV Protease Activity Assay kit is a fluorogenic-based assay to measure TEV protease activity. \nThe kit contains a TEV protease substrate that is labeled with fluorophore FAM and a quencher. \nProteolytic activity of TEV protease cleaves the substrate and releases the FAM, resulting in the \nproduction of bright fluorescence which can be measured using a fluorescence reader at ex\/em of 490 \nnM\/520 nm. TEV protease activity then can be calculated in accordance with the fluorescence intensity. \nPurified TEV protease is included in the kit as a positive control.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eMeasure TEV protease activity.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA microplate reader capable of measuring fluorescence intensity is required. Aurora Biolabs, LLC; www.aurorabiolabs.com; San Diego, CA, USA. Tel: 858-215-4510 or 858-374-6010; Tech: 858-453-5700 58-453-5700\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e190001B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e96-well microplate, black\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Dilute 1 mM 5-FAM to 20 µM with the assay buffer prepared at step A (assay buffer A).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Make 2-fold series of dilutions with the assay buffer a to get 10, 5, 2.5 1.25, 0.625, 0.3125 and 0 µM solutions.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Aliquot 50 µL of the diluted solution to each well (96-well plate).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Dilute substrate solution 25-fold with the assay buffer A.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Add 50 µl of diluted substrate to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Measure fluorescent intensity at excitation of 490 nm and emission of 520 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Use the same machine settings when measure TEV protease activity afterwards. 5-FAM Standard y t i s n e t n I e c n e c s e r o u F l 10000 8000 6000 4000 2000 0 0 2000 4000 6000 8000 10000 FAM [nM] C. Measure TEV protease positive control activity\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Thaw TEV protease protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: TEV protease protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Dilute the TEV protein 125-fold with the assay buffer A (from 1000 ng\/µL to 16 ng\/µL). Then, make a further dilution to 8, 4, 2, 0.5, 0 ng\/µL.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Add 50 µl of diluted protein solution to each well (Test amount of the protein will be 400, 200, 100, 50, 25 and 0 ng per reaction).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 11.\u003c\/strong\u003e Dilute substrate solution 25-fold with assay buffer A.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 12.\u003c\/strong\u003e Add 50 µl of diluted substrate to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 13.\u003c\/strong\u003e Incubate at room temperature for 1 hour.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 14.\u003c\/strong\u003e Measure fluorescent intensity at excitation of 490 nm and emission of 520 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 15.\u003c\/strong\u003e Plot fluorescent intensity versus protein concentration on a graph as below (subtract the average fluorescent intensity readings in the 0 ng wells from all of other wells to remove fluorescence background). TEV Activity y t i s n e t n I e c n e c s e r o u F l 7000 6000 5000 4000 3000 2000 1000 0 0 100 200 TEV [ng] 300 400 D. Measure TEV protease activity\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 16.\u003c\/strong\u003e Dilute TEV protease protein to 8, 4, 2, 0.5, 0 ng\/µL with the assay buffer A.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 17.\u003c\/strong\u003e Add 50 µl of diluted protein solution to each well (Test amount of the protein will be 400, 200, 100, 50, 25 and 0 ng per reaction). We recommend to run the reactions in duplicate.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 18.\u003c\/strong\u003e Dilute substrate solution 25-fold with assay buffer A.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 19.\u003c\/strong\u003e Add 50 µl of diluted substrate to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 20.\u003c\/strong\u003e Incubate at room temperature for 1 hour.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 21.\u003c\/strong\u003e Measure fluorescent intensity at excitation of 490 nm and emission of 520 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 22.\u003c\/strong\u003e Plot fluorescent intensity versus protein concentration on a graph as below (subtract the average fluorescent intensity readings in the 0 ng wells from all of other wells to remove fluorescence background).\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eProteolysis (affinity tag removal; protein engineering)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eGeneral \/ Biotechnology\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"96 reactions","offer_id":53238302277997,"sku":"190001AK","price":599.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12c-nucleotide-exchange-assay-kit-bht20700012","title":"Kras G12C Nucleotide Exchange Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \nsignaling pathways. The Ras signaling pathway plays an important role in cell proliferation and \ndifferentiation. Conversion of Kras from the inactive GDP-bound state to the active GTP-bound state \ntriggers the downstream effector and promotes cell growth. RAS genes are frequently mutated in \nvarious human tumors. These mutations block the GTPase activity of RAS and lock RAS in the GTP-\nbound state, resulting in constitutively active signals through the downstream cascades leading to \ncancer cell proliferation.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12C) nucleotide exchange assay is a TR-FRET based assay. The assay kit is designed to \ndetect the GTP binding status of Kras (G12C) in the presence of SOS1, the most-studied guanine \nnucleotide exchange factor (GEF) of Kras. The Tag2-Kras in this assay kit is recognized by a Terbium-\nlabeled anti-Tag2 antibody (HTRF donor). If Kras binds to a fluorescence-labeled GTP (HTRF \nacceptor), the donor and the acceptor will be brought in close proximity. Excitation of Terbium (340 nm) \ngenerates fluorescence resonance energy transfer (FRET) to the fluorescence-labeled GTP acceptor, \nwhich consequently fluoresces at 665 nm (figure below). Thus, GTP binding to Kras can be quantitively \nmeasured by calculation of the fluorescent ratio of 665 nm\/620 nm. The inhibitor blocking the nucleotide \nexchange will reduce the HTRF signal. \n\nAurora Biolabs LLC, San Diego, CA 92121; www.aurorabiolabs.com; \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit Kras activation for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-NK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e24 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302441837,"sku":"5727-4122NK","price":1699.0,"currency_code":"USD","in_stock":false}]},{"product_id":"wee1-binding-assay-kit-bht20700031","title":"WEE1 Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eWEE1, a nuclear kinase, belongs to WEE kinase family that negatively regulates the cell cycle \n\nvia phosphorylation of CDK1. WEE1 serves as a dual-specificity kinase which selectively \nphosphorylates both Thr14 and Tyr15 residues of both CDK1 and CDK2 to restrain their activation and \nhalt cell cycle progression in the response to DNA damage. Overexpression of WEE1 is commonly \nobserved in malignant cells and its high expression has been associated with poor rates of survival in \nvarious cancer types. Inhibition of WEE1 facilitates or even expedites mitotic progression, leading to \nan increase in genomic instability. Therefore, WEE1 is considered a potential therapeutic target for \ncancer treatment.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe WEE1 binding assay kit is a TR-FRET based assay, which is designed to screen compounds that \n\nbind to WEE1. If the WEE1 with N-terminal tag2 binds to a fluorescence-labelled tracer (fluorescent \n\nreceptor, emission at 665 mm), it brings the Terbium (fluorescence donor, emission at 620 mm) \n\nconjugated with anti-Tag2 antibody close to the fluorescent acceptor. Activation of the Terbium results \n\nin fluorescence resonance energy transfer (FRET), and leads to the receptor fluorescent emission at \n\n665 mm. The competitive binding of a non-fluorescence-labeled compound will reduce the receptor \n\nsignal. Thus, the compound binding status can be quantitively measured by calculating the ratio of the \n\nemission fluorescence intensity of the acceptor (665 nm) and donor (620 nm). \n\n Aurora Biolabs LLC, San Diego, CA, USA. www.aurorabiolabs.com; \n\n 1 \n\n \n \n \n \n \n \n \n \n \n \n \n\nReference\nLOt \n\nMatheson, J.C., et al., Trends Pharmacol Sci. 2016 Oct;37(10):872-881.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit WEE1 activity for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e759331-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e20 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare WEE1 solution Thaw WEE1 protein on ice. Upon first thaw, briefly spin the tube to recover all of the contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: WEE1 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not store and re-use the diluted protein. Dilute the WEE1 protein 120-fold (1 µL WEE1 + 119 µL 1X assay buffer containing DTT). Add 8 µl of diluted protein solution to each positive control well and inhibitor test well. Add 8 µl of 1X DTT containing buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare fluorescence-labeled tracer and Tb-labeled anti-Tag2 antibody solution Thaw the tracer and the antibody to room temperature. Dilute the tracer 50-fold and the antibody 200-fold with 1X assay buffer containing DTT. For example, add 4 µl of the tracer and 1 µl of the anti-tag2 antibody to 200 µl of 1X DTT containing assay buffer.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Incubate the reaction at room temperature for 60 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eCell Cycle Regulation (DNA damage response; CDK1\/CDK2 inhibition)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology (overexpressed in malignant cells)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302376301,"sku":"759331BK","price":1999.0,"currency_code":"USD","in_stock":false}]},{"product_id":"caspase-3-activity-assay-kit-bht20700032","title":"Caspase-3 Activity Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eCaspase-3 is a critical enzyme involved in the process of apoptosis, or programmed cell death. It’s \nconsidered one of the “effector caspases,” which are the final executioners of the apoptotic process. \nCaspase-3 is activated in response to various cellular stress signals and, once activated, it leads to the \nbreakdown of cellular components and the death of the cell. This ensures that damaged or unwanted \ncells are efficiently removed without causing harm to the surrounding tissue. In the context of cancer, \nthe role of caspase-3 is significant because cancer cells often evade apoptosis, allowing them to survive \nand proliferate uncontrollably. The dysregulation of apoptosis is a hallmark of cancer, and as a result, \nmany cancer cells have reduced or altered caspase-3 activity. Because of its essential role in apoptosis, \ncaspase-3 has been studied in the context of cancer (where apoptosis may be disrupted) and \nneurodegenerative diseases (where increased apoptosis can occur).\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eCaspase-3 activity assay kit is designed to measure caspase-3 activity for enzyme profiling and inhibitor \nscreening . Proteolytic activity of caspase-3 cuts the fluorogenic substrate and releases the fluorophore, \nresulting in fluorescent intensity increase which can be measured using a microplate reader at \nexcitation at 360 nm and emission at 460 nm.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eQuantification of caspase-3 activity and High throughput screening of compounds that have effects on \nthe enzyme activity for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA microplate reader capable of measuring fluorescence intensity at excitation at 360 nm and emission at 460 nm.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e2x Caspase assay buffer\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eMaterials needed but not supplied\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare a series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare caspase-3 solution Thaw caspase-3 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: caspase-3 protein is sensitive to freeze\/thaw cycles. Limit the number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the caspase-3 protein 500-fold (1 µL caspase-3 + 499 µL 1X DTT-containing assay buffer). Add 8 µl of diluted protein solution to each positive control wells and inhibitor test wells. Add 8 µl of 1X DTT containing buffer to each of the negative control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor solution Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each negative and positive control wells. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare caspase-3 substrate Thaw the substrate at room temperature. Dilute the substrate 50-fold (1 µL of 1 mM tracer + 49 µL 1X DTT-containing assay buffer). Add 10 µl of diluted substrate to each well. Dilute enough substrate for single use. Store remaining undiluted tracer at -80°C. Do not re-use the diluted tracer.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Incubate the reaction at room temperature for 60 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Measure fluorescent intensity Fluorescent intensity should be measured by excitation wavelength at 360 nm and emission at 460 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e Caspase3 Activity\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e0.0006 uM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eApoptosis (Caspase-3 execution pathway)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology; Neurodegenerative Disease\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302409069,"sku":"810030","price":499.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12d-nucleotide-exchange-assay-kit-bht20700015","title":"Kras G12D Nucleotide Exchange Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \nsignaling pathways. The Ras signaling pathway plays an important role in cell proliferation and \ndifferentiation. Conversion of Kras from the inactive GDP-bound state to the active GTP-bound state \ntriggers the downstream effector and promotes cell growth. RAS genes are frequently mutated in \nvarious human tumors. These mutations block the GTPase activity of RAS and lock RAS in the GTP-\nbound state, resulting in constitutively active signals through the downstream cascades leading to \ncancer cell proliferation.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12D) nucleotide exchange assay is a TR-FRET based assay. The assay kit is designed to \ndetect the GTP binding status of Kras (G12D) in the presence of SOS1, the most-studied guanine \nnucleotide exchange factor (GEF) of Kras. The Tag2-Kras in this assay kit is recognized by a Terbium-\nlabeled anti-Tag2 antibody (HTRF donor). If Kras binds to a fluorescence-labeled GTP (HTRF \nacceptor), the donor and the acceptor will be brought in close proximity. Excitation of Terbium (340 nm) \ngenerates fluorescence resonance energy transfer (FRET) to the fluorescence-labeled GTP acceptor, \nwhich consequently fluoresces at 665 nm (figure below). Thus, GTP binding to Kras can be quantitively \nmeasured by calculation of the fluorescent ratio of 665 nm\/620 nm. The inhibitor blocking the nucleotide \nexchange will reduce the HTRF signal. \n\n Aurora Biolabs LLC, San Diego, CA 92121; www.aurorabiolabs.com; \n\nKras (G12D) Nucleotide Exchange Assay Kit\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit Kras activation for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-NK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare 1X assay buffer containing 1 mM DTT (1X DTT-containing assay buffer) For example, mix 996 µl distilled water with 1000 µl of 2X assay Buffer (Catalogue number: 5727- NK-B) and 4 µl of 0.5 M DTT. Make only enough 1X DTT-containing assay buffer as needed for the assay. Store the remaining 2X assay buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare SOS1 solution Thaw SOS1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: SOS1 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the SOS1 protein 400-fold (1 µL SOS1 + 399 µL 1X DTT-containing assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of 1X DTT-containing assay buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare Kras solution Kras (G12D) Nucleotide Exchange Assay Kit Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein to 440-fold (1µL Kras G12D + 439 µL 1X DTT-containing assay buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:200 and dilute fluorescence-labeled GTP 1:40 in 1X DTT-containing assay buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 5 µl of fluorescence-labeled GTP + 194 µl of 1X DTT-containing assay buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 20 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e21 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302474605,"sku":"5727-4123NK","price":1699.0,"currency_code":"USD","in_stock":false}]},{"product_id":"sars-cov-2-nucleocapsid-protein-binding-kit-for-mouse-antibody-bht20700028","title":"SARS-CoV-2 Nucleocapsid Protein Binding Kit (For mouse antibody)","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eSARS-CoV-2 Nucleocapsid protein (NP) is one of the core components of SARS-CoV-2 virus. It forms \na complex with viral genomic RNA in a helical symmetrical structure and plays a key role in the process \nof virus replication and assembly. Since NP is abundantly expressed during infection, it can be used \nas an important diagnostic marker for COVID-19 and also can be used as a potential drug target or \ndeveloping vaccines.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe SARS-CoV-2 Nucleocapsid protein (NP) Binding kit is a TR-FRET based assay, that is designed \nto detect binding status of NP to the test antibody. Terbium-labeled anti-Tag5 antibody serves as \nfluorescence donor, that binds to the Tag5-NP. If a test mouse antibody binds to NP, fluorescence-\nlabeled anti-mouse antibody (fluorescence acceptor) will be brought in close proximity with the \nfluorescence donor. Excitation of Terbium (340 nm) generates fluorescence resonance energy transfer \n(FRET) to the fluorescence-labeled acceptor, which consequently fluoresces at 665 nm (figure below). \nThus, the test antibody binding to NP can be quantitively measured by calculation of the fluorescent \nratio of 665 nm\/620 nm.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of antibodies that bind to NP.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required. Aurora Biolabs LLC, San Diego, CA 92121, USA; www.aurorabiolabs.com; SARS-CoV-2 Nucleocapsid Protein Binding Kit (For mouse antibody)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e2x Assay Buffer\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003eCustomer Test anti-NP-mouse antibody (to be tested antibody)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare SARS-CoV-2 Nucleocapsid protein Dilute SARS-CoV-2 Nucleocapsid protein (NP) 1,500-fold with 1X DTT-containing assay buffer. For example: 1 µl of NP + 1,499 µl of 1X DTT-containing assay buffer. Add 5 µl of diluted NP protein to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare Antibody solution Prepare mouse antibody with 1X DTT-containing assay buffer to the concentration to be tested. Add 5 µl of diluted antibody solution to each well except negative control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare dye solution\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Incubate the reaction at room temperature for 1 hour.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample signal. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% activity. The \npercent activity in the presence of each compound is calculated according to the following \n\nAurora Biolabs LLC, San Diego, CA 92121, USA; www. SARS-CoV-2 Nucleocapsid Protein Binding Kit \n(For mouse antibody) equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence of the \ncompound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e Nucleocapsid Protein Binding\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eViral Antigen Recognition; Vaccine Response\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eInfectious Disease (COVID-19)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302572909,"sku":"728263","price":1799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g13d-nucleotide-exchange-assay-kit-bht20700023","title":"Kras G13D Nucleotide Exchange Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \nsignaling pathways. The Ras signaling pathway plays an important role in cell proliferation and \ndifferentiation. Conversion of Kras from the inactive GDP-bound state to the active GTP-bound state \ntriggers the downstream effector and promotes cell growth. RAS genes are frequently mutated in \nvarious human tumors. These mutations block the GTPase activity of RAS and lock RAS in the GTP-\nbound state, resulting in constitutively active signals through the downstream cascades leading to \ncancer cell proliferation.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G13D) nucleotide exchange assay is a TR-FRET based assay. The assay kit is designed to \ndetect the GTP binding status of Kras mutant (G13D). The Tag2-Kras (G13D) in this assay kit is \nrecognized by a Terbium-labeled anti-Tag2 antibody (HTRF donor). If Kras binds to a fluorescence-\nlabeled GTP (HTRF acceptor), the donor and the acceptor will be brought in close proximity. Excitation \nof Terbium (340 nm) generates fluorescence resonance energy transfer (FRET) to the fluorescence-\nlabeled GTP acceptor, which consequently fluoresces at 665 nm (figure below). Thus, GTP binding to \nKras can be quantitively measured by calculation of the fluorescent ratio of 665 nm\/620 nm. The \ninhibitor blocking the nucleotide exchange will reduce the HTRF signal.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit Kras activation for drug discovery. \n\n Aurora Biolabs LLC, San Diego, CA 92121; www.aurorabiolabs.com; \n\n 1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-NK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare 1X assay buffer containing 1 mM DTT (1X DTT-containing assay buffer) For example, mix 996 µl distilled water with 1000 µl of 2X assay Buffer (Catalogue number: 5727- NK-B) and 4 µl of 0.5 M DTT. Make only enough 1X DTT-containing assay buffer as needed for the assay. Store the remaining 2X assay buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare Kras solution Thaw Kras (G13D) protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein to 800-fold (1 µL Kras G13D + 799 µL 1X assay buffer containing DTT). Add 8 µl of diluted protein solution to the positive control and inhibitor test wells. Add 8 µl of 1X assay buffer containing DTT) to the negative control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:200 and dilute fluorescence-labeled GTP 1:40 in 1X DTT-containing assay buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 5 µl of fluorescence-labeled GTP + 194 µl of 1X DTT-containing assay buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm. Negative Control Positive Control Inhibitor Test\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e Kras (G13D) Nucleotide Exchange Activity\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302736749,"sku":"5727-4133NK","price":1699.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12d-craf-binding-assay-kit-bht20700013","title":"Kras G12D–cRAF Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \n\nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \n\nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \n\ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \n\nhuman tumors, the Ras-RAF signaling pathway is considered a potential therapeutic target for cancer \n\ntreatment.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12D)-cRAF binding assay kit is a TR-FRET based assay, which is designed to detect the \n\nbinding status between Kras and cRAF. Tag2-Kras (G12D) in this assay kit is loaded with GppNHp, \n\nwhich represents the activated Kras. The Ras binding domain (RBD) of cRAF has a Tag1 at N-terminus. \n\nA Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a fluorescence donor (HTRF \n\ndonor), activation of which results in fluorescence resonance energy transfer (FRET) if Tag1-cRAF \n\nbinds to the Kras, since the binding brings Terbium on the anti-Tag2 antibody close to the fluorophore \n\non the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status can be quantitively measured by \n\ncalculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 \n\nnm). Blocking the Kras-cRAF binding will reduce the HTRF signal. \n\n-4510 or 858453-5700 Fax: 855-898-3979 \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (G12D) and \ncRAF for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-BK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in Binding buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in Binding buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in Binding buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare cRAF solution Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 480-fold (1 µL cRAF + 479 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of DTT containing Binding buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare Kras (G12D) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. -4510 or 858453-5700 Fax: 855-898-3979 3 Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein 340-fold (1µL Kras G12D + 339 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody and fluorescence-labeled anti-Tag1 antibody 1:200 in DTT containing Binding buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 1 µl of fluorescence-labeled anti-Tag1 antibody + 198 µl of DTT containing Binding buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e Kras (G12D)-cRAF Binding\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e7.3 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eConditions:\u003c\/em\u003e 35 nM cRAF\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302638445,"sku":"5727-4123BK","price":1799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g13d-craf-cypa-inhibitor-assay-kit-bht20700022","title":"Kras G13D\/cRAF\/CYPA\/Inhibitor Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \nhuman tumors, the Ras-RAF signaling pathway is considered an important therapeutic target for cancer \n\ntreatment. However, Ras is considered undruggable since it lacks suitable binding pockets on the \nsurface. Recently, a discovery of a small molecule inhibitor blocks Ras-RAF signaling pathway by \n\nremolding Cyclophilin A (CYPA) and forming a CYPA:drug:KRAS ternary complex. This inhibitory \nstrategy provides a new method for developing drugs targeting Kras for treatment of cancers.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G13D) Inhibitor assay kit is a TR-FRET based assay, which is designed to screen Kras \n\ninhibitors and determine the Kras-inhibitor binding affinity. Tag2-Kras (G13D) in this assay kit is loaded \nwith GppNHp, which represents the activated Kras. The Ras binding domain (RBD) of cRAF in the kit \nhas a Tag1 at N-terminus. A Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a \nfluorescence donor (HTRF donor), activation of which results in fluorescence resonance energy \n\ntransfer (FRET) if Tag1-cRAF binds to the Kras, since the binding brings Terbium on the anti-Tag2 \nantibody close to the fluorophore on the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status \ncan be quantitively measured by calculating the ratio of the emission fluorescence intensity of the \nacceptor (665 nm) and donor (620 nm). If an inhibitor associated with CYPA binds to the Kras and \nblocks the cRAF binding, the HTRF signal will be reduced. \n\n-4510 or 858453-5700 Fax: 855-898-3979 \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (G13D) and \ncRAF for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-CK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000, Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare compound dilution buffer containing 2 mM DTT (CD buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare 1X Assay Buffer containing 2 mM DTT (AB buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare Kras (G13D) solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare cRAF solution\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate sample HTRF signal of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in CD buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in CD buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in CD buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare 1X Assay Buffer containing 2 mM DTT (AB buffer) For example, mix 500 µl of 2X Kras Binding Buffer, 496 µl of distilled water and 4 µl of 0.5 M DTT. Make only enough AB buffer as needed for the assay. Store the remaining Binding buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare Kras (G13D) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein 135-fold (1µL Kras G13D + 134 µL AB buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of AB buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of CD buffer to each of negative and positive control well. -4510 or 858453-5700 Fax: 855-898-3979 3\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare cRAF solution Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 400-fold (1 µL cRAF + 399 µL of AB buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody and fluorescence-labeled anti-Tag1 antibody 1:200 in AB buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 1 µl of fluorescence-labeled anti-Tag1 antibody + 198 µl of AB buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e103 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eReference Compound:\u003c\/em\u003e RMC-6236\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302671213,"sku":"5727-4133CK","price":1999.0,"currency_code":"USD","in_stock":false}]},{"product_id":"pkmyt1-binding-assay-kit-bht20700030","title":"PKMYT1 Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003ePKMYT1, a membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase, belongs to \nWEE kinase family that plays an important role in the regulation of mitosis. PKMYT1 is involved in cell \ncycle progression and in response to DNA damages by inhibition of CDK1 activity through specific \nphosphorylation of Tyr15 and Thr14. Overexpression of PKMYT1 is observed in both solid and \nhematological tumors. Therefore, PKMYT1 is considered a potential therapeutic target for cancer \ntreatment.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe PKMYT1 binding assay kit is a TR-FRET based assay, which is designed to screen compounds \nthat bind to PKMYT1. A fluorescence-labelled tracer, that can bind to PKMYT1, and the N-terminal \nGST-tagged full-length human PKMYT1 are used in this assay kit. A Terbium-labeled anti-GST \nantibody binding to the GST-PKMYT1 serves as a fluorescence donor (HTRF donor). if the \nfluorescence-labeled tracer binds to the PKMYT1, the binding brings Terbium on the anti-GST antibody \nclose to the fluorophore on the tracer (HTRF acceptor). Activation of the Terbium results in fluorescence \nresonance energy transfer (FRET). Thus, the binding status can be quantitively measured by \ncalculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 \nnm). The competitive binding of a non-fluorescence-labeled compound will reduce the FRET signal.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the PKMYT1 activity. \n\n Aurora Biolabs, LLC; www.aurorabiolabs.com; \nSan Diego, CA, USA. Tel: 858-215-4510 or 858-453-5700 Fax: 855-898-3979 \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e756981-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e20 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare assay buffer containing 1 mM DTT\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare PKMYT1 solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare fluorescence-labeled tracer and Tb-labeled anti-Tag2 antibody solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 60 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate the ratio of the fluorescent intensity of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare PKMYT1 solution Thaw PKMYT1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: PKMYT1 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the PKMYT1 protein 20-fold (1 µL PKMYT1 + 19 µL 1X assay buffer containing DTT). Add 8 µl of diluted protein solution to each positive control well and inhibitor test well. Add 8 µl of 1X DTT containing buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare fluorescence-labeled tracer and Tb-labeled anti-Tag2 antibody solution Thaw the tracer and the antibody to room temperature. Dilute the tracer 100-fold and the antibody 200-fold with 1X assay buffer containing DTT. For example, add 2 µl of the tracer and 1 µl of the anti-tag2 antibody to 200 µl of 1X DTT containing assay buffer.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Incubate the reaction at room temperature for 60 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eCell Cycle Regulation (Mitosis entry; CDK1 inhibition)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology (solid and hematological tumors)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302605677,"sku":"756981BK","price":1999.0,"currency_code":"USD","in_stock":false}]},{"product_id":"sars-cov-2-mpro-3cl-protease-assay-kit-bht20700026","title":"SARS-CoV-2 Mpro (3CL Protease) Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eSARS-CoV-2 Mpro Assay Kit \n Mpro of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2 Mpro, also referred to as SARS-CoV-\n\n2 Main protease, SARS-CoV-2 3CL protease) plays an essential role in viral replication by processing the \n\npolyproteins 1ab at 11 cleavage sites. Inhibiting the activity of this enzyme would block viral replication, \n\nmaking it a promising target for anti-coronaviral therapeutic agents. \n\nDescription \n\nThe Aurora SARS-CoV-2 Mpro assay kit is a homogeneous FRET-based assay for screening Mpro inhibitors. \n\nThe assay is fast and convenient, and requires just two \nsteps. In the first step, the Mpro enzyme \nis \npreincubated with the compound for 30 minutes. The \nreaction is initiated by adding substrate solution at \nthe second step. Fluorescent intensity is measured \nwith a fluorescent plate reader at the excitation \nemission \nwavelengths of 340-360 nm \nwavelengths of 460-480 nm. \n\nand \n\nItem \n2X Protease Assay Buffer \n0.5 M DTT \n5 mM Substrate \nRecombinant SARS-CoV-2 Mpro \nBlack low binding 96 well plate \n\nAmount \n20 ml \n200 µl \n10 µl \n5 µg \n1 \n\nStorage \n-20°C \n-20°C \n-80°C \n-80°C \nRT \n\nMaterials supplied \n\nCatalogue Number \n728205 \n\n728202 \n728206 \n\nMaterials Needed but not supplied \n\nA microplate reader capable of measuring fluorescence at excitation wavelengths of 340-360 nm and \nemission wavelengths of 460-480 nm. \n\nStability \n\n12 months if stored under the indicated conditions.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution. If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 5 µl to the 50 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare Mpro solution. Thaw Mpro enzyme on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Mpro enzyme is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted enzyme. Dilute the Mpro enzyme to 5 ng\/µl in 1X assay buffer. Add 20 µl of diluted enzyme solution to each of positive control well and inhibitor test well. Add 1X buffer to each of background well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add the inhibitor solution Add 5 µl of 1X assay buffer to each background well and positive control well if the inhibitor is diluted in 1X buffer. Add 5 µl of 1X assay buffer with 10% DMSO to each of background well and positive control well if the inhibitor is diluted in 1X assay buffer with 10% DMSO. Add 5 µl of diluted inhibitor solution from Step 2 to each of the inhibitor test well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Incubate at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare substrate solution During the incubation of the enzyme and the inhibitor solution, dilute the 5 mM substrate solution to 20 µM in 1X assay buffer. Make only enough solution as need for the assay. Store the remaining 5 mM Substrate solution to -80°C. Add 25 µl of diluted substrate solution to each of well, including background wells, positive control wells and the inhibitor test wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate at room temperature for 2 hours.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure the fluorescent intensity Measure the fluorescent intensity at the excitation wavelengths of 340-360 nm and the emission wavelengths of 460-480 nm\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate percentage activity of the enzyme \n\n% Activity= \n\n(Fp – Fb) – (Fi-Fb) \nFp - Fb \n\nX 100 \n\nWhere Fp refers to Fluorescent intensity of the positive control, Fb refers to Fluorescent intensity of \nbackground, and Fi refers to Fluorescent intensity of the inhibitor. Graph the percentage activity as a function of the inhibitor concentration to determine the IC50 of the test \ninhibitor. No CPD refers to no compound control \n(compound vehicle control).\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eViral Polyprotein Processing \/ Replication\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eInfectious Disease (COVID-19)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"96 reactions","offer_id":53238302507373,"sku":"728203","price":899.0,"currency_code":"USD","in_stock":false}]},{"product_id":"tr-fret-parp2-trapping-assay-kit-bht20700025","title":"TR-FRET PARP2 Trapping Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003ePARP2 (Poly (ADP-ribose) polymerase 2) is a member of the PARP family and plays a crucial role in \nDNA repair, particularly in the repair of single-strand breaks (SSBs) in DNA. It binds to DNA at the site \nof damage, becomes catalytically activated, and uses NAD⁺ as a substrate to add poly (ADP-ribose) \n(PAR) chains to itself and other proteins—a process called PARylation that results in the recruitment \nof other DNA repair proteins to the damaged site. Because of the high negative charge of PAR \npolymers, extensive autoPARylation of PARP2 leads to the dissociation of PARP2 from DNA, which is \nrequired for DNA repair completion. PARP2 is often overexpressed in various cancers, including breast, \novarian, prostate, lung, and glioblastoma. This overexpression is thought to support tumor cell survival. \nSome PARP inhibitors not only block the catalytic activity of PARP2 but also trap PARP2 on DNA at \nsites of damage, preventing its release. This creates a toxic DNA-protein complex that interferes with \nDNA replication and repair, leading to cell death, particularly in cancer cells deficient in homologous \nrecombination repair (e.g., BRCA1\/2-mutant cells).\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe TR-FRET PARP2 Trapping Assay Kit is designed to detect the poly-ADP-ribosylation activity of \nPARP2 and the status of PARP2 trapping on DNA. The DNA substrate in the kit is labeled with a \nfluorophore (acceptor). A Terbium (Tb)-labeled anti-Tag2 antibody that binds to Tag2-Kras serves as \nthe fluorescence donor. Activation of Tb results in fluorescence resonance energy transfer (FRET) if \nPARP2 binds to the fluorescence-labeled DNA, since the binding brings the fluorescence donor into \nclose proximity with the fluorophore acceptor. Thus, the binding status can be quantitatively measured \nby calculating the ratio of the emission fluorescence intensities of the acceptor (665 nm) and the donor \n(620 nm). In the presence of NAD⁺, auto-PARylation of PARP2 leads to its dissociation from DNA, \nresulting in a decrease in the FRET signal. Inhibition of auto-PARylation activity traps PARP2 on the \nDNA, and the FRET signal remains high.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the auto-PARylation activity of PARP2 for drug \ndiscovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e7277-TA-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare PARP2 solution Thaw PARP2 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: PARP2 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the PARP2 protein 500-fold (1 µL PARP2 + 499 µL assay buffer). Add 4 µl of diluted protein solution to each of positive control wells and inhibitor test wells. Add 4 µl of assay buffer to each of negative control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare the DNA substrate solution Dilute the fluorescence-labeled DNA 20-fold (1 µL DNA + 19 µL assay buffer). Add 4 µl of the diluted DNA solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare NAD+ solution Dilute the NAD+ 25-fold (1 µL NAD+ + 24 µL assay buffer). Add 5 µl of diluted NAD+ solution to each of positive control and compound test wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:100. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 99 µl assay buffer. Add 5 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e0.7 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eDNA Damage Response (DDR)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology (breast; ovarian; prostate)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302540141,"sku":"72772TAK","price":1799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"tr-fret-vhl-binding-assay-kit-384-bht20700033","title":"TR-FRET VHL Binding Assay Kit (384)","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eVon Hippel–Lindau (VHL) is a member of an E3 ubiquitin ligase, a five-component complex including \nVHL, Cullin 2 (CUL2), Elongin B, Elongin C and RBX1 (RING-box protein 1). It is one of the most widely \nused E3 ligase recruiters in the design of PROTACs (Proteolysis-Targeting Chimeras) for targeted \nprotein degradation (TPD) drug discovery. VHL plays a critical role in bringing the target protein and \n\nthe ubiquitination machinery together for protein degradation via the proteasome.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe TR-FRET VHL Binding Assy kit is designed to measure the binding affinity of VHL and its ligand, \nand it includes Tag1-VHL-5C (VHL\/CUL2\/EloC\/EloB\/RBX1 complex), Terbium-labeled Anti-Tag1 \nantibody and fluorescent labeled VHL ligand VH032. The binding of VHL to the ligand brings Terbium \n(fluorescence donor) on the anti-Tag1 antibody in close proximity to the fluorophore (FL) on VH032 \n(fluorescent receptor), which results in fluorescence resonance energy transfer (FRET). Thus, the \nbinding status of VHL and VH032 can be quantitively determined using HTRF signal by calculating the \nratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 nm). If an \ncompound binds to the VHL and blocks VH032 binding, the HTRF signal will be reduced.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that bind to VHL for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required. -4510 or 858453-5700 Fax: 855-898-3979 1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e845225-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-5C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eFluorescence-labeled VH032 (FL-VH032)\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e20 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare VHL-5C solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare FL-VH032 solution\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 60 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate sample HTRF signal of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare VHL-5C solution Thaw VHL-5C protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: VHL-5C protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the VHL-5C protein 40-fold (1µL VHL-5C + 39 µL assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of assay buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of assay buffer to each of negative and positive control wells. If the compound is diluted in 10% DMSO, add 2 µl of assay buffer containing 10% DMSO to each of\tnegative and positive control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare FL-VH032 solution Dilute FL-VH032 10-fold (1 µL FL-VH032 + 9 µL of assay buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag1 antibody 1:100 in assay buffer. For example: 1 µl of Terbium- labeled anti-Tag1 antibody + 99 µl of assay buffer. Add 10 µl of this dye mixture to each well. -4510 or 858453-5700 Fax: 855-898-3979 3\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Incubate the reaction at room temperature for 60 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e TR-FRET VHL Binding Assay\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eReference Compound:\u003c\/em\u003e VH032\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eUbiquitin-Proteasome \/ Targeted Protein Degradation\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology; PROTAC Drug Discovery\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302703981,"sku":"845225","price":1299.0,"currency_code":"USD","in_stock":false}]},{"product_id":"ox40-ox40l-inhibitor-binding-assay-kit-bht20700002","title":"OX40\/OX40L Inhibitor Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eOX40 (CD134; TNFRSF4) is a T-cell co-stimulatory receptor that belong to the tumor necrosis factor \nreceptor superfamily (TNFRSF). Binding of OX40 to OX40L (CD252, CD134L, TNFSF4) triggers signal \ntransduction pathways to activate immune response and regulate T-cell activation, proliferation, \ndifferentiation, expansion, and survival. OX40 agonists are being developed and tested in clinical trials \nfor cancer treatment. OX40 antagonists, by inhibiting OX40, can dampen T cell activity, potentially \nreducing inflammation and autoimmune responses. Modulating the OX40\/OX40L pathway have \nimplications for treating inflammatory and autoimmune diseases, as well as cancer immunotherapy.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe OX40-OX40L binding assay kit is a TR-FRET based assay, which is designed to detect the binding \nstatus between OX40 and OX40L. Tag6-OX40 and Tag7-OX40L are included in this assay kit. Binding \nof Tag6-OX40 to Tag7-OX40L brings the Terbium (Tb, HTRF donor) and the fluorophore d2 (HTRF \nacceptor) in a proximity distance, and activation of Tb results in fluorescence resonance energy transfer \n(FRET). Thus, the binding status can be quantitively measured by calculating the ratio of the emission \nfluorescence intensity of the acceptor (665 nm) and donor (620 nm). Interference of the OX40-OX40L \nbinding will reduce the HTRF signal. \n\nOX40\/OX40L Inhibitor Binding Assay Kit\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between OX40 and OX40L for drug \ndiscovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eAssay buffer\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare OX40 solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare OX40L solution\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 1 hour.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate sample HTRF signal of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare OX40 solution Thaw OX40 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: OX40 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the OX4L protein 80-fold (1 µL OX40L + 79 µL 1X assay buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare OX40L solution Thaw OX40 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: OX40L protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the OX40L protein 600-fold (1 µL OX40L + 599 µL 1X assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of assay buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare dye solution OX40\/OX40L Inhibitor Binding Assay Kit Dilute Terbium-labeled anti-Tag6 antibody and fluorescence-labeled anti-Tag7 antibody 1:200 in assay buffer. For example: 1 µl of Terbium-labeled anti-Tag6 antibody + 1 µl of fluorescence- labeled anti-Tag7 antibody + 198 µl of assay buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Incubate the reaction at room temperature for 1 hour.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \n\n4 µl \n\n2 µl \n\n6 µl \n\n4 µl \n\n10 µl \n\n20 µl\n following equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e OX40-OX40L Binding Activity\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e0.00028 uM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eImmune Co-stimulatory Pathway (T cell activation)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology \/ Immunotherapy\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302769517,"sku":"2369401","price":1699.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12c-craf-binding-assay-kit-bht20700010","title":"Kras G12C–cRAF Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \n\nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \n\nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \n\ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \n\nhuman tumors, the Ras-RAF signaling pathway is considered a potential therapeutic target for cancer \n\ntreatment.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12C)-cRAF binding assay kit is a TR-FRET based assay, which is designed to detect the \n\nbinding status between Kras and cRAF. Tag2-Kras (G12C) in this assay kit is loaded with GppNHp, \n\nwhich represents the activated Kras. The Ras binding domain (RBD) of cRAF has a Tag1 at N-terminus. \n\nA Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a fluorescence donor (HTRF \n\ndonor), activation of which results in fluorescence resonance energy transfer (FRET) if Tag1-cRAF \n\nbinds to the Kras, since the binding brings Terbium on the anti-Tag2 antibody close to the fluorophore \n\non the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status can be quantitively measured by \n\ncalculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 \n\nnm). Blocking the Kras-cRAF binding will reduce the HTRF signal. \n\n-4510 or 858453-5700 Fax: 855-898-3979 \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (G12C) and \ncRAF for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-BK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in Binding buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in Binding buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in Binding buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare cRAF solution Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 480-fold (1 µL cRAF + 479 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of DTT containing Binding buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare Kras (G12C) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. -4510 or 858453-5700 Fax: 855-898-3979 3 Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein 340-fold (1µL Kras G12C + 339 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody and fluorescence-labeled anti-Tag1 antibody 1:200 in DTT containing Binding buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 1 µl of fluorescence-labeled anti-Tag1 antibody + 198 µl of DTT containing Binding buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e Kras (G12D)-cRAF Binding\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e7.3 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eConditions:\u003c\/em\u003e 35 nM cRAF\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302802285,"sku":"5727-4122BK","price":1799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"papain-like-plpro-protease-assay-kit-bht20700027","title":"Papain-like (PLpro) Protease Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eSARS-CoV-2 PLpro Assay Kit \n PLpro of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2 PLpro, also referred to as SARS-CoV-\n2 Papain-like protease, Nsp3) plays an essential role in processing the polyproteins 1ab for viral replication. \nInhibition of this enzyme activity would block viral replication, making it a promising target for anti-\ncoronaviral therapeutic agents. \n\nDescription \n\nThe Aurora SARS-CoV-2 PLpro assay kit is a homogeneous assay for screening PLpro Inhibitors. \n\nThe assay is fast and convenient, and requires just \ntwo steps. In the first step, the PLpro enzyme is \npreincubated with the compound for 30 minutes. \nThe reaction is initiated by adding substrate \nsolution at the second step. Fluorescent intensity is \nmeasured with a fluorescent plate reader at the \nexcitation wavelengths of 340-360 nm and \nemission wavelengths of 460-480 nm. \n\nMaterials supplied \n\nCatalogue Number \n\nItem \n\nAmount \n\nStorage \n\n728205 \n\n728252 \n\n728256 \n\n2X Protease Assay Buffer \n\n0.5 M DTT \n\n5 mM Substrate \n\nRecombinant SARS-CoV-2 PLpro \n\nBlack low binding 96 well plate \n\n20 ml \n\n200 µl \n\n10 µl \n\n5 µg \n\n1 \n\n-20°C \n\n-20°C \n\n-80°C \n\n-80°C \n\nRT \n\nMaterials Needed but not supplied \n\nA microplate reader capable of measuring fluorescence at excitation wavelengths of 340-360 nm and \nemission wavelengths of 460-480 nm. \n\nStability \n\n12 months if stored under the indicated conditions.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution. If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 5 µl to the 50 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare PLpro solution. Thaw PLpro enzyme on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: PLpro enzyme is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted enzyme. Dilute the PLpro enzyme to 1.25 ng\/µl in 1X assay buffer. Add 20 µl of diluted enzyme solution to each of positive control well and inhibitor test well. Add 1X assay buffer to each of background well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add the inhibitor solution Add 5 µl of 1X assay buffer to each background well and positive control well if the inhibitor is diluted in 1X buffer. Add 5 µl of 1X assay buffer with 10% DMSO to each of background well and positive control well if the inhibitor is diluted in 1X buffer with 10% DMSO. Add 5 µl of diluted inhibitor solution from Step 2 to each of the inhibitor test well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Incubate at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare substrate solution During the incubation of the enzyme and the inhibitor solution, dilute the 5 mM substrate solution to 20µM in 1X assay buffer. Make only enough solution as need for the assay. Store the remaining 5 mM Substrate solution to -80°C. Add 25 µl of diluted substrate solution to each of well, including background wells, positive control wells and the inhibitor test wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate at room temperature for 2 hours.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure the fluorescent intensity\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate percentage activity of the enzyme \n\n% Activity= \n\n(Fp – Fb) – (Fi-Fb) \nFp - Fb \n\nX 100 \n\nWhere Fp refers to Fluorescent intensity of the positive control, Fb refers to Fluorescent intensity of \nbackground, and Fi refers to Fluorescent intensity of the inhibitor. Graph the percentage activity as a function of the inhibitor concentration to determine the IC50 of the test \ninhibitor. No CPD refers to no compound control \n(compound vehicle control).\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"96 reactions","offer_id":53238302835053,"sku":"728253","price":825.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12r-nucleotide-exchange-assay-kit-bht20700017","title":"Kras G12R Nucleotide Exchange Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \nsignaling pathways. The Ras signaling pathway plays an important role in cell proliferation and \ndifferentiation. Conversion of Kras from the inactive GDP-bound state to the active GTP-bound state \ntriggers the downstream effector and promotes cell growth. RAS genes are frequently mutated in \nvarious human tumors. These mutations block the GTPase activity of RAS and lock RAS in the GTP-\nbound state, resulting in constitutively active signals through the downstream cascades leading to \ncancer cell proliferation.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12R) nucleotide exchange assay is a TR-FRET based assay. The assay kit is designed to \ndetect the GTP binding status of Kras (G12R) in the presence of SOS1, the most-studied guanine \nnucleotide exchange factor (GEF) of Kras. The Tag2-Kras in this assay kit is recognized by a Terbium-\nlabeled anti-Tag2 antibody (HTRF donor). If Kras binds to a fluorescence-labeled GTP (HTRF \nacceptor), the donor and the acceptor will be brought in close proximity. Excitation of Terbium (340 nm) \ngenerates fluorescence resonance energy transfer (FRET) to the fluorescence-labeled GTP acceptor, \nwhich consequently fluoresces at 665 nm (figure below). Thus, GTP binding to Kras can be quantitively \nmeasured by calculation of the fluorescent ratio of 665 nm\/620 nm. The inhibitor blocking the nucleotide \nexchange will reduce the HTRF signal. \n\nAurora Biolabs LLC, San Diego, CA 92121, USA; www.aurorabiolabs.com; \n\nKras (G12R) Nucleotide Exchange Assay Kit\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit Kras activation for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-NK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e20 µL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003e1. Prepare 1X assay buffer containing 1 mM DTT (1X DTT-containing assay buffer)\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution\u003c\/li\u003e\n\u003cli\u003ePrepare SOS1 solution\u003c\/li\u003e\n\u003cli\u003eAdd inhibitor\u003c\/li\u003e\n\u003cli\u003ePrepare Kras solution\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate the reaction at room temperature for 20 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure fluorescent intensity\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli\u003eExcitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003cli\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well.\u003c\/li\u003e\n\u003cli\u003eCalculate percentage activity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare 1X assay buffer containing 1 mM DTT (1X DTT-containing assay buffer) For example, mix 996 µl distilled water with 1000 µl of 2X assay Buffer (Catalogue number: 5727- NK-B) and 4 µl of 0.5 M DTT. Make only enough 1X DTT-containing assay buffer as needed for the assay. Store the remaining 2X assay buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare SOS1 solution Thaw SOS1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: SOS1 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the SOS1 protein 80-fold (10 µL SOS1 + 790 µL 1X DTT-containing assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of 1X DTT-containing assay buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare Kras solution Kras (G12R) Nucleotide Exchange Assay Kit Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein to 380-fold (1µL Kras G12R + 379 µL 1X DTT-containing assay buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:200 and dilute fluorescence-labeled GTP 1:40 in 1X DTT-containing assay buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 5 µl of fluorescence-labeled GTP + 194 µl of 1X DTT-containing assay buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 20 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e55 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302867821,"sku":"5727-4127NK","price":1699.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12v-nucleotide-exchange-assay-kit-bht20700020","title":"Kras G12V Nucleotide Exchange Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \n\nsignaling pathways. The Ras signaling pathway plays an important role in cell proliferation and \ndifferentiation. Conversion of Kras from the inactive GDP-bound state to the active GTP-bound state \n\ntriggers the downstream effector and promotes cell growth. RAS genes are frequently mutated in \nvarious human tumors. These mutations block the GTPase activity of RAS and lock RAS in the GTP-\n\nbound state, resulting in constitutively active signals through the downstream cascades leading to \ncancer cell proliferation.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12V) nucleotide exchange assay is a TR-FRET based assay. The assay kit is designed to \n\ndetect the GTP binding status of wild type Kras in the presence of SOS1, the most-studied guanine \n\nnucleotide exchange factor (GEF) of Kras. The Tag2-Kras in this assay kit is recognized by a Terbium-\n\nlabeled anti-Tag2 antibody (HTRF donor). If Kras binds to a fluorescence-labeled GTP (HTRF \n\nacceptor), the donor and the acceptor will be brought in close proximity. Excitation of Terbium (340 nm) \n\ngenerates fluorescence resonance energy transfer (FRET) to the fluorescence-labeled GTP acceptor, \n\nwhich consequently fluoresces at 665 nm (figure below). Thus, GTP binding to Kras can be quantitively \n\nmeasured by calculation of the fluorescent ratio of 665 nm\/620 nm. The inhibitor blocking the nucleotide \n\nexchange will reduce the HTRF signal. \n\nAurora Biolabs LLC, San Diego, CA 92121; www.aurorabiolabs.com; \n\nKras (G12V) Nucleotide Exchange Assay Kit \n\n LOt\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit Kras activation for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-NK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare 1X aasay buffer containing 1 mM DTT (1X DTT-containing assay buffer) For example, mix 996 µl distilled water with 1000 µl of 2X assay Buffer (catalogue number: 5727- NK-B) and 4 µl of 0.5 M DTT. Make only enough 1X DTT-containing assay buffer as needed for the assay. Store the remaining 2X assay buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare SOS1 solution Thaw SOS1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: SOS1 protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the SOS1 protein 1,000-fold (1 µL SOS1 + 999 µL 1X DTT-containing assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of 1X DTT-containing assay buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare Kras solution\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:200 and dilute fluorescence-labeled GTP 1:40 in 1X DTT-containing assay buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 5 µl of fluorescence-labeled GTP + 194 µl of 1X DTT-containing assay buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 20 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e23 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238303031661,"sku":"5727-4128NK","price":1699.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12v-craf-binding-assay-kit-bht20700018","title":"Kras G12V–cRAF Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \n\nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \n\nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \n\ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \n\nhuman tumors, the Ras-RAF signaling pathway is considered a potential therapeutic target for cancer \n\ntreatment.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12V)-cRAF binding assay kit is a TR-FRET based assay, which is designed to detect the \n\nbinding status between Kras and cRAF. Tag2-Kras (G12V) in this assay kit is loaded with GppNHp, \n\nwhich represents the activated Kras. The Ras binding domain (RBD) of cRAF has a Tag1 at N-terminus. \n\nA Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a fluorescence donor (HTRF \n\ndonor), activation of which results in fluorescence resonance energy transfer (FRET) if the Tag1-cRAF \n\nbinds to Kras, since the binding brings Terbium on the anti-Tag2 antibody close to the fluorophore on \n\nthe anti-Tag1 antibody (HTRF acceptor). Thus, the binding status can be quantitively measured by \n\ncalculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 \n\nnm). Blocking the Kras-cRAF binding will reduce the HTRF signal. \n\nAurora Biolabs, 10052 Mesa Ridge Court, Suite 103, San Diego, CA 92121, USA; www.aurorabiolabs.com; \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (G12V) and \ncRAF for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required. Amount Storage\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eCatalog number\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in Binding buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in Binging buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in Binding buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare cRAF solution Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 480-fold (1 µL cRAF + 479 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of DTT containing Binding buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare Kras (G12V) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:200 and dilute fluorescence-labeled anti-Tag1 antibody 1:40 in DTT containing Binding buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 5 µl of fluorescence-labeled anti-Tag1 antibody + 194 µl of DTT containing Binding buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302900589,"sku":"5727-4128BK","price":1799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g13d-craf-binding-assay-kit-bht20700021","title":"Kras G13D–cRAF Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \n\nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \n\nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \n\ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \n\nhuman tumors, the Ras-RAF signaling pathway is considered a potential therapeutic target for cancer \n\ntreatment.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G13D)-cRAF binding assay kit is a TR-FRET based assay, which is designed to detect the \n\nbinding status between Kras and cRAF. Tag2-Kras (G13D) in this assay kit is loaded with GppNHp, \n\nwhich represents the activated Kras. The Ras binding domain (RBD) of cRAF has a Tag1 at N-terminus. \n\nA Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a fluorescence donor (HTRF \n\ndonor), activation of which results in fluorescence resonance energy transfer (FRET) if Tag1-cRAF \n\nbinds to the Kras, since the binding brings Terbium on the anti-Tag2 antibody close to the fluorophore \n\non the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status can be quantitively measured by \n\ncalculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 \n\nnm). Blocking the Kras-cRAF binding will reduce the HTRF signal. \n\n-4510 or 858453-5700 Fax: 855-898-3979 \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (G13D) and \ncRAF for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-BK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in Binding buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in Binding buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in Binding buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare cRAF solution Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number of freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 400-fold (1 µL cRAF + 399 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of DTT containing Binding buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare Kras (G13D) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. -4510 or 858453-5700 Fax: 855-898-3979 3 Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein 670-fold (1 µL Kras G13D + 669 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each positive control and compound test wells. Add 4 µl of DTT containing Binding buffer to each of negative control wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody and fluorescence-labeled anti-Tag1 antibody 1:200 in DTT containing Binding buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 1 µl of fluorescence-labeled anti-Tag1 antibody + 198 µl of DTT containing Binding buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eAssay:\u003c\/em\u003e Kras (G13D)-cRAF Binding\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302933357,"sku":"5727-4133BK","price":1799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"t7-high-yield-rna-synthesis-kit-bht20700035","title":"T7 High Yield RNA Synthesis Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eBacteriophage\t T7\t RNA\t Polymerase\t is\t a\t 99\t kDa\t protein\t that\t recognizes\t T7\t phage\t promoters\t with\t high\t\nspecificity\t and\t subsequently\t initiates\t transcription.\t T7\t RNA\t polymerase\t is\t a\t single\t subunit,\t highly\t\nprocessive\tand\tstable\tenzyme,\tcharacteristics\tthat\tmake\tit\tsuitable\tfor\ta\tbroad\trange\tof\tbiochemistry\tand\t\nmolecular\tbiology\tapplications.\t\n\nDescription \n\nThe\tAurora\tT7\tRNA\tPolymerase\tIn\tVitro\tTranscription\tKit\tis\ta\tquick\tand\teasy\tapproach\tto\tgenerate\tlarge\t\namounts\t of\t RNA\t in\t vitro.\t The\t RNA\t product\t from\t the\t kit\t is\t suitable\t for\t RNA\t structural,\t functional,\t and\t\nenzymatic\t(ie.\tribozyme)\tstudies,\tproduction\tof\tRNA\tprobes\tfor\thybridization\tblotting\tor\tRNase\tprotection\t\nassays,\tRNA\tvaccine\tproduction,\tmicroarray\tand\tmicroinjection,\tanti-sense\tRNA\tand\tRNAi\texperiments,\t\nand\tin\tvitro\ttranslation.\t\t\n\nThe\tassay\tis\tfast\tand\tconvenient,\tand\trequires\tthe\tT7\tRNA\tpolymerase,\tNTP\tmix\t(UTP,\tATP,\t\nCTP,\tand\tGTP),\treaction\tbuffer,\tand\ta\tsuitable\tDNA\ttemplate.\tThe\tmodified\tnucleotide\tN1-\nMethyl-Pseudo\tUTP\tis\tincorporated\tin\tour\tT7\tRNA\tPolymerase\tIn\tVitro\tTranscription\tKit-II.\t\n\nFigure\t1\tillustrates\tthe\tT7\ttranscription\twith\tT7\tpromoter\tsequence\tand\tthe\ttranscription\tstart\t\nsite.\t\n\nT7 Promoter \n\n+1 (Transcription Start Site) \n\n5’ TAATACGACTCACTATAGGG \n\n3’ ATTATGCTGAGTGATATCCC \n\nRNA Transcript \n\n5’ GGG \n\nT7\tTranscription\t\n\n3’ \n\n5’ \n\n3’ \n\nFigure 1. T7 RNA transcription \n\nMaterials Supplied \nCatalogue No. \nK777627-E\t\nK777627-B\t\nK777627-A\t\nK777627-G\t\nK777627-C\t\nK777627-U\t\nK777627-T\t\nK777627-H\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eT7 RNA Polymerase MIX\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e0.5\tug\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eStability\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Set\tup\treactions\tfor\tRNA\tsynthesis 1) Thaw\tthe\tkit\tcomponents\ton\tice\tand\tbriefly\tspin\tthe\ttubes\tto\trecover\tthe\tfull\tcontents\tat\tthe bottom\tof\tthe\ttube.\tFor\tT7\tRNA\tpolymerase,\tmake\taliquots\tof\tthe\tenzyme\tfor\tsingle\tuse.\tStore remaining\taliquot\tprotein\tat\t-80°C. 2) Assemble\tthe\treaction\tat\troom\ttemperature\tin\tthe\tfollowing\torder:\u003c\/li\u003e\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cp\u003eKb\n\n20\n\n40\n\n60\n\n80\n\n100\n\n120\n\n140\n\nReaction\tTime,\tMinutes\t\n\nRelated\tproducts:\t\nCatalog\t# \n5727-4121NK \n5727-4122NK \n5727-4123NK \n5727-4133NK \n5727-4127NK \n5727-4128NK \n5727-4121BK \n5727-4122BK \n5727-4123BK \n5727-4127BK \n5727-4128BK \n\nProduct\tName \nKras WT Nucleotide Exchange Assay Kit \nKras G12C Nucleotide Exchange Assay Kit \nKras G12D Nucleotide Exchange Assay Kit \nKras G13D Nucleotide Exchange Assay Kit \nKras G12R Nucleotide Exchange Assay Kit \nKras G12V Nucleotide Exchange Assay Kit \nKras WT–cRAF Binding Assay Kit \nKras G12C–cRAF Binding Assay Kit \nKras G12D–cRAF Binding Assay Kit \nKras G12R–cRAF Binding Assay Kit \nKras G12V–cRAF Binding Assay Kit \n\n728203 \n728253 \n728263 \n728273 \n\n362101 \n\nK777627 \n756981BK \n\n759331BK \n\n34343BK \n810030 \n\n910010 \n\nSARS-CoV-2 Mpro (3CL Protease) Assay Kit \nSARS-CoV-2 Papain-like Protease Assay Kit \nSARS-CoV-2 Nucleocapsid Protein Binding Kit (For mouse antibody) \nSARS-CoV-2 Nucleocapsid Protein Binding Kit (For rabbit antibody) \n\nDNA Polymerase Theta Activity Assay Kit \nT7 High Yield RNA Synthesis Kit \n\nPKMYT1 Binding Assay Kit \n\nWEE1 Binding Assay Kit \n\neIF4E\/eIF4G Binding Assay Kit \n\nCaspase-3 Activity Assay Kit \n\nIDO1 Activity Assay Kit for Inhibitor Screening \n\nSize \n384 reactions \n384 reactions \n384 reactions \n384 reactions \n384 reactions \n384 reactions \n384 reactions \n384 reactions \n384 reactions \n384 reactions \n384 reactions \n\n96 reactions \n96 reactions \n384 reactions \n384 reactions \n\n96 reactions, 384 rec \n25, 50, 100 reactions \n\n384 reactions \n\n384 reactions \n\n384 reactions \n384 reactions \n\n96 reactions \n\n \n\n3 \n\n\t\n\t\n \n \n \n \n \n \n\t\n \n\t\n\n T7 High Yield RNA Synthesis Kit \n\n 5727-4122G \n\nKras G12C, GST-tag \nKras G12D, GST-tag, GDP Loaded \n\n5727-4123G-G \n5727-4128G-GP Kras G12V, GST-tag, GppNHp loaded \n7671 \n7671HA \n7237231 \n\nSOS1, No tag \nSOS1, His-Avi-tagged \nHuman RBD-RAF1, N-His tag, C-FLAG tag \n\n180001 \n180002 \n180003 \n\n12-0009MH \n12-0009H \n12-0009M \n12-0009 \n\n12-0010 \n12-0011 \n12-0012 \n\n7657643 \n7657283 \n\nC352E1-10 \nC352A2-10 \n\n225201-1 \n62581 \n\n232340 \n2323405 \n2323155 \n236940 \n2369405 \n2344875 \n237351 \n56781 \n52352-FL \n5756981-FL \n\n5756981-CDD \n\n5756981-CDP \n\nRecombinant Human Malic enzyme 1 (ME1) \nRecombinant Human Malic enzyme 2 (ME2) \nRecombinant Human Malic enzyme 3 (ME3) \n\nRecombinant His-tagged Human MNK2 D228G \nRecombinant His-tagged Human MNK2 \nRecombinant Human MNK2 D228G \nRecombinant Human MNK2 \n\nHis-tagged Human eIF4E \nHis-tagged Human eIF4E Complexed With EIF4G \nHis-tagged Human eIF4E Complexed With m7GTP \nDNA Polymerase Theta-N-Helicase Domain \nDNA Polymerase Theta-C terminal Domain \n\nGST-CDK2:his-CyclinE1 \nGST-CDK2:His-CyclinA2 \n\nRecombinant Human BCL2 \n\nHuman MALT1 (caspase-IG3) \nRecombinant Human CD40 \nRecombinant Human CD40L \nRecombinant Human CD155 \nRecombinant Human OX40 \nRecombinant Human OX40L \nRecombinant Human GITRL \nRecombinant Human PD-L1 \n\nRecombinant Full-length Human MST1 \nRecombinant Human CDK2 \nRecombinant Human Full Length PKMYT1 \nRecombinant Human PKMYT1, catalytic domain – \ndephosphorylated \nRecombinant Human PKMYT1, catalytic domain – \nphosphorylated \n\n50 µg, 100 µg \n50 µg, 100 µg \n50 µg, 100 µg \n100 µg, 1 mg \n100 µg, 1 mg \n100 µg \n\n10 μg, 100 µg, 500 µg, 1 mg \n10 μg, 100 µg, 500 µg, 1 mg \n10 μg, 100 µg, 500 µg, 1 mg \n\n10 µg, 100 µg \n10 µg, 100 µg \n10 µg, 100 µg \n10 µg, 100 µg \n\n50 µg, 100 µg \n50 µg, 100 µg \n50 µg, 100 µg \n\n20 µg, 100 µg \n20 µg, 100 µg \n\n10 µg \n10 µg \n100 µg \n\n20 µg \n\n100 µg \n100 µg \n100 µg \n100 µg \n100 µg \n100 µg \n100 µg \n10 µg, 50 µg, 100 µg, 500 µg \n50 µg, 500 µg \n10 µg, 50 µg, 100 µg, 500 µg \n\n10 µg , 50 µg, 100 µg, 500 µg \n\n10 µg , 50 µg, 100 µg, 500 µg \n\nProducts\tare\tfor\tresearch\tuse\tonly\tand\tare\tnot\tintended\tfor\thuman\tuse.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eIn Vitro Transcription\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eRNA Therapeutics; Gene Therapy; Vaccine; General\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"25 reactions","offer_id":53238302966125,"sku":"K777627-25","price":110.0,"currency_code":"USD","in_stock":false},{"title":"50 reactions","offer_id":53238305489261,"sku":"K777627-50","price":199.0,"currency_code":"USD","in_stock":false},{"title":"100 reactions","offer_id":53238305522029,"sku":"K777627-100","price":399.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-wt-craf-cypa-inhibitor-assay-kit-bht20700008","title":"Kras WT\/cRAF\/CYPA\/Inhibitor Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \n\nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \n\nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \n\ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \n\nhuman tumors, the Ras-RAF signaling pathway is considered an important therapeutic target for cancer \n\ntreatment. However, Ras is considered undruggable since it lacks suitable binding pockets on the \n\nsurface. Recently, a discovery of a small molecule inhibitor blocks Ras-RAF signaling pathway by \n\nremolding Cyclophilin A (CYPA) and forming a CYPA:drug:KRAS ternary complex. This inhibitory \n\nstrategy provides a new method for developing drugs targeting Kras for treatment of cancers.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (WT) Inhibitor assay kit is a TR-FRET based assay, which is designed to screen Kras \n\ninhibitors and determine the Kras-inhibitor binding affinity. Tag2-Kras (WT) in this assay kit is loaded \n\nwith GppNHp, which represents the activated Kras. The Ras binding domain (RBD) of cRAF in the kit \n\nhas a Tag1 at N-terminus. A Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a \n\nfluorescence donor (HTRF donor), activation of which results in fluorescence resonance energy \n\ntransfer (FRET) if Tag1-cRAF binds to the Kras, since the binding brings Terbium on the anti-Tag2 \n\nantibody close to the fluorophore on the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status \n\ncan be quantitively measured by calculating the ratio of the emission fluorescence intensity of the \n\nacceptor (665 nm) and donor (620 nm). If an inhibitor associated with CYPA binds to the Kras and \n\nblocks the cRAF binding, the HTRF signal will be reduced. \n\n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (WT) and cRAF \nfor drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-CK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000, Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in CD buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in CD buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in CD buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare 1X Assay Buffer containing 2 mM DTT (AB buffer) For example, mix 500 µl of 2X Kras Binding Buffer, 496 µl of distilled water and 4 µl of 0.5 M DTT. Make only enough AB buffer as needed for the assay. Store the remaining Binding buffer at -20°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Prepare Kras (WT) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein 110-fold (1µL Kras WT + 109 µL AB buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of AB buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of CD buffer to each of negative and positive control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare cRAF solution 3 Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 400-fold (1 µL cRAF + 399 µL of AB buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody and fluorescence-labeled anti-Tag1 antibody 1:200 in AB buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 1 µl of fluorescence-labeled anti-Tag1 antibody + 198 µl of AB buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 10.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate sample HTRF signal of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e101 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eReference Compound:\u003c\/em\u003e RMC-6236\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eRAS-RAF Signaling Pathway\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology (KRAS-driven cancers)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238302998893,"sku":"5727-4121CK","price":1999.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-g12r-craf-binding-assay-kit-bht20700016","title":"Kras G12R–cRAF Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \n\nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \nproliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the \n\nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \n\ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \nhuman tumors, the Ras-RAF signaling pathway is considered a potential therapeutic target for cancer \n\ntreatment.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (G12R)-cRAF binding assay kit is a TR-FRET based assay, which is designed to detect the \n\nbinding status between Kras and cRAF. Tag2-Kras (G12R) in this assay kit is loaded with GppNHp, \n\nwhich represents the activated Kras. The Ras binding domain (RBD) of cRAF has a Tag1 at N-terminus. \n\nA Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a fluorescence donor (HTRF \n\ndonor), activation of which results in fluorescence resonance energy transfer (FRET) if Tag1-cRAF \n\nbinds to Kras, since the binding brings Terbium on the anti-Tag2 antibody close to the fluorophore on \n\nthe anti-Tag1 antibody (HTRF acceptor). Thus, the binding status can be quantitively measured by \n\ncalculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 \n\nnm). Blocking the Kras-cRAF binding will reduce the HTRF signal. \n\nAurora Biolabs, 10052 Mesa Ridge Court, Suite 103, San Diego, CA 92121, USA; www.aurorabiolabs.com;\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (G12R) and \ncRAF for drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-BK-B\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e5727-4122-T2P\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e20 µL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-80°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eTerbium-labeled anti-Tag2 antibody\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e20 µL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-80°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in Binding buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in Binding buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in Binding buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare cRAF solution Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 480-fold (1 µL cRAF + 479 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of DTT containing Binding buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare Kras (G12R) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:200 and dilute fluorescence-labeled anti-Tag1 antibody 1:40 in DTT containing Binding buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 5 µl of fluorescence-labeled anti-Tag1 antibody + 194 µl of DTT containing Binding buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238303064429,"sku":"5727-4127BK","price":1799.0,"currency_code":"USD","in_stock":false}]},{"product_id":"kras-wt-craf-binding-assay-kit-bht20700007","title":"Kras WT–cRAF Binding Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eKras is a member of the RAS protein family, which are a class of small GTPases involved in cell \n\nsignaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell \n\nproliferation, differentiation and survival. Conversion of Ras from the inactive GDP-bound state to the \n\nactive GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key \n\ndownstream effector of RAS. Since the frequently mutated Ras genes are associated with various \n\nhuman tumors, the Ras-RAF signaling pathway is considered a potential therapeutic target for cancer \n\ntreatment.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Principle\u003c\/h4\u003e\n\u003cp\u003eThe Kras (WT, wild type)-cRAF binding assay kit is a TR-FRET based assay, which is designed to \n\ndetect the binding status between Kras and cRAF. Tag2-Kras (WT) in this assay kit is loaded with \n\nGppNHp, which represents the activated Kras. The Ras binding domain (RBD) of cRAF has a Tag1 at \n\nN-terminus. A Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a fluorescence \n\ndonor (HTRF donor), activation of which results in fluorescence resonance energy transfer (FRET) if \n\nthe Tag1-cRAF binds to Kras, since the binding brings Terbium on the anti-Tag2 antibody close to the \n\nfluorophore on the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status can be quantitively \n\nmeasured by calculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and \n\ndonor (620 nm). Blocking the Kras-cRAF binding will reduce the HTRF signal. The ratio of the emission \n\nfluorescence intensity of the acceptor (665 nm) and donor (620 nm).\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eApplication\u003c\/h4\u003e\n\u003cp\u003eHigh throughput screening of compounds that inhibit the binding between activated Kras (WT) and cRAF \nfor drug discovery.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eInstrument Required\u003c\/h4\u003e\n\u003cp\u003eA HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eKit Components\u003c\/h4\u003e\n\u003ctable class=\"bhc-spec-table\" style=\"width:100%;border-collapse:collapse;font-size:0.85em\"\u003e\n\u003cthead\u003e\u003ctr style=\"background:#1a5c58;color:#fff\"\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eCatalog No.\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px;text-align:left\"\u003eItem\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eAmount\u003c\/th\u003e\n\u003cth style=\"padding:4px 8px\"\u003eStorage\u003c\/th\u003e\n\u003c\/tr\u003e\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003eCatalog number\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e25 mL\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e-20°C\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px\"\u003e384-well microplate, White\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003e\u003c\/td\u003e\n\u003ctd style=\"padding:4px 8px;text-align:center\"\u003eRoom temperature\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003eMicroplate reader, HTRF® certified microplate reader (such as Tecan M1000 or Tecan Spark, etc.)\u003c\/li\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in Binding buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in Binding buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in Binding buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare cRAF solution Thaw cRAF protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: cRAF protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 480-fold (1 µL cRAF + 479 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of DTT containing Binding buffer to each of negative control well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each negative and positive control well. Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Prepare Kras (WT) solution Thaw Kras protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Kras protein is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein to 110-fold (1µL Kras WT + 109 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:200 and dilute fluorescence-labeled anti-Tag1 antibody 1:40 in DTT containing Binding buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 5 µl of fluorescence-labeled anti-Tag1 antibody + 194 µl DTT containing Binding buffer. Add 10 µl of this dye mixture to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the reaction at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 620 nm.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Excitation wavelength at 340 nm and emission at 665 nm.\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 1 — Calculate HTRF Signal\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003eHTRF = (Fluorescence at 665 nm \/ Fluorescence at 620 nm) × 10,000\u003c\/code\u003e\n\u003c\/div\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate the HTRF signal (ratio of the fluorescent intensity at 665 mm\/620 mm) of each well. Calculate percentage activity \n\nIn the absence of the compound (positive control), the sample signal (P) is defined as 100% \nactivity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% \nactivity. The percent activity in the presence of each compound is calculated according to the \nfollowing equation: % activity = (S-N)\/(P-N) X100, where S= the sample signal in the presence \nof the compound.\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"384 reactions","offer_id":53238303097197,"sku":"5727-4121BK","price":1799.0,"currency_code":"USD","in_stock":false}]}],"url":"https:\/\/www.ebiohippo.com\/collections\/aurora-biolabs.oembed","provider":"BioHippo","version":"1.0","type":"link"}