Wehavedevelopedahigh-throughputassayformeasuringE2thioesterformationthatcanbeusedeitherasasecondaryscreentodeconvoluteE3assay“hits”orasaprimaryscreenforinhibitorsofE2thioesterformation.UbiquitinthioesterformationontheE2conjugatingenzymebringsadonorfluorophoreontheubiquitinincloseproximitytotheacceptorfluorophoreassociatedwiththeE2toallowFRETandanincreasedfluorescencesignalinproportiontotheamountofubiquitinthioester.Thelong-livedfluorescenceoftheterbiumdonorallowsatime-delayedreADIngoftheFRETsignal,ortime-resolvedFRET(TR-FRET),whichreducesthecontributionofshort-livedbackgroundemissionsresultingfrombuffers,proteins,andchemicalcompounds.TheassayisconfiguredwiththeE1activatingenzymeUBE1(UBA1)andtheE2conjugatingenzymeUBE2D3(UbcH5c).Theassaycanberunina½-volume96-wellor384-wellformatandproducesareproducIBLesignalwithaZ’>0.7.PleaseinquireaboutdevelopmentofanassayforyourE2ofinterest.1kitcontainssufficientreagentsfor1.5x1/2-volume96-wellplates.

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