{"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}],"url":"https:\/\/www.ebiohippo.com\/products\/eif4e-eif4g-binding-assay-kit-bht20700005","provider":"BioHippo","version":"1.0","type":"link"}