ASSAYPROTOCOL:
Materials:SIRTAssayBuffer(BPS#50090)SIRT5Substrate(#50126)SIRT5enzyme(#50016)orotherSIRT5-containingsampleNUNCblack96-wellplate,lowproteinbinding(VWR#62408-936).SIRTAssayDeveloper(BPS#50089)
Step1:Setup50μlofSirt5reactionscontainingSIRTassaybuffer,400ngSIRT5enzyme,and20μMSIRT5substrateinthe96-wellplate.Incubateat37ºCfor30min.
Step2:Add50μlofSIRTAssayDeveloper(2x)(BPS#50089)toeachwellandincubatetheplateatroomtemperaturefor15min.
Step3:Readsampleinamicrotiter-platereADIngfluorimetercapableofexcitationatawavelengthintherange350-380nmanddetectionofemittedlightintherange440-460nm.“Blank”value(noenzymecontrol)issubtractedfromallothervalues.
Stableatleast6monthsfromdateofreceipt,whenstoredasdirectedat-20°Cor-80°C
1.Madsen,A.S.andOlsen,C.A.,J.Med.Chem.2012,55:5582-5590.
2.Du,J.,etal.,Science,11November2011,334:806-809.