{"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}],"url":"https:\/\/www.ebiohippo.com\/products\/kras-g12v-nucleotide-exchange-assay-kit-bht20700020","provider":"BioHippo","version":"1.0","type":"link"}