{"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}],"url":"https:\/\/www.ebiohippo.com\/products\/wee1-binding-assay-kit-bht20700031","provider":"BioHippo","version":"1.0","type":"link"}