{"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}],"url":"https:\/\/www.ebiohippo.com\/products\/kras-g12d-craf-cypa-inhibitor-assay-kit-bht20700014","provider":"BioHippo","version":"1.0","type":"link"}