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NovagenUSAandCanadaEuropeAllOtherCountriesTel(800)[e-mail]0800126461GermanyFreecall08001003496IrelandTollFree1800409445UnitedKingdomFreephone0800622935A[e-mail]————————————————[e-mail]————————————————®TransmembraneProteinExtractionKitTableofContentsAbouttheKits........................................................................................................2Description2Components2Storage2Equipmentandmaterialsrequiredbutnotsupplied3ProteoExtract®TransmembraneProteinExtractionProtocol..................................3Extractionofmembraneproteinsfromadherentculturedcells3Extractionofmembraneproteinsfromsuspensioncellsorfrozencellpellets4Extractionofmembraneproteinsfromtissue6FrequentlyAskedQuestions...................................................................................8Appendix...............................................................................................................8Exampleextractions8ExamplesoftotalproteinyieldsusingTM-PEK9©2009EMDChemicalsInc.,anaffiliateofMerckKGaA,Darmstadt,Germany.Allrightsreserved.TheNovagen®logoandNovagen®nameareregisteredtrademarksofEMDChemicalsInc.intheUnitedStatesandincertainotherjurisdictions.ProteoExtract®isaregisteredtrademarkofMerckKGaA,Darmstadt,Germany.TRITON®isaregisteredtrademarkofDowChemicalCompany.Cat.No.PageUSAandCanadaTel(800)[e-mail][e-mail][e-mail]@novagen.comUserProtocolTB477Rev.B0409Page2of9AbouttheKitsProteoExtract®TransmembraneProteinExtractionKit1kit71772-3DescriptionTheProteoExtract®TransmembraneProteinExtractionKit(TM-PEK)usesanovel,detergent-freechemistryforextractionofperipherally-associatedandintegralmembraneproteins,suchasG-ProteinCoupledReceptors(GPCRs),frommammaliancellsandtissues.Themembraneproteinfractionisdirectlycompatiblewithenzymeassays,nativeanddenaturinggelelectrophoresis,Westernblotting,immunoprecipitation,and(followingin-geltrypticdigestion)massspectrometry.TheTM-PEKmethodcomprisesatwo-stepprotocolfortheenrichmentoftransmembrane(TM)proteins.Inthefirststep,cellsorhomogenizedtissuesarepermeablizedusingExtractionBuffer1andtheirsoluble(cytoplasmic)fractionseparatedfromtheinsoluble(membrane)fractionbycentrifugation.Inthesecondstep,membraneproteinsareextractedfromthelipidbilayerusingoneoftwonovelextractionbuffers.Thesebuffers,ExtractionBuffer2AandExtractionBuffer2B,arepreparedbydilutingTM-PEKReagentAorTM-PEKReagentBintoExtractionBuffer2.Theoptimalextractionreagentmustbedeterminedempirically,asitwilldependoncharacteristicsoftheprotein(s)ofinterestandintendeddownstreamapplications.ExtractionBuffer2Aisaverymildextractionagent,allowingforrecoveryoffragileproteincomplexes.ExtractionBuffer2B,bycomparison,isahighlyefficientextractionagentandfacilitatesrecoveryofdifficult-to-extracttransmembraneproteins,includingthosewithmultipletransmembranesegments.IfusingtheTM-PEKkitforthefirsttime,prepareduplicatesamples.ExtractthefirstsetofreplicateswithTM-PEKReagentA,diluted2-foldwithExtractionBuffer2.ExtractthesecondsetofreplicateswithTM-PEKReagentB,diluted2-foldwithExtractionBuffer2.ExtractionconditionscanbeoptimizedfurtherbyvaryingthedilutionrangeofeachreagentintoExtractionBuffer,fromundilutedtoa10-folddilution.Unlikealternativemembraneproteinextractionmethods,theTM-PEKkitdoesnotrequiresonication,rigorousvortexing,time-consumingultracentrifugation,orincubationatelevatedtemperatures.Theabsenceofsuchharshtreatmentsminimizespotentialdamageorchangestothetargetprotein.EachTM-PEKkitprovidessufficientreagentstoprocessatotalof40samples(20samplesusingExtractionBuffer2Aattherecommendeddilution,and20samplesusingExtractionBuffer2Battherecommendeddilution).Eachsamplemaybecomprisedof1–5x107culturedcellsor25–50mgoftissueComponents•••••40mlExtractionBuffer150mlExtractionBuffer22.0mlTM-PEKReagentA2.0mlTM-PEKReagentB0.4mlProteaseInhibitorCocktailSetIIIStorageStoreallcomponentsat4°Cforupto6months.Forprolongedstorage,dispensethecomponentsinworkingaliquotsandstoreat–20°C.Beforeperformingextractions,thawallkitcomponentsatroomtemperatureandmixcompletely.Avoidrepeatedfreezingandthawing.NotethattheproteaseinhibitorsareprovidedinDMSOandmustbekeptatroomtemperatureduringtheextractionproceduretopreventfreezing.Cat.No.PageUSAandCanadaTel(800)[e-mail][e-mail][e-mail]@novagen.comUserProtocolTB477Rev.B0409Page3of9Equipmentandmaterialsrequiredbutnotsupplied•••••Rockingplatformorellipticalmixer(requiredatroomtemperature