BCA蛋白定量试剂盒(Thermo)使用指南

INSTRUCTIONSPierceBiotechnologyPOBox117(815)968-0747(815)968-7316faxNumberDescription23225PierceBCAProteinAssayKit,sufficientreagentsfor500test-tubeor5000microplateassays23227PierceBCAProteinAssayKit,sufficientreagentsfor250test-tubeor2500microplateassaysKitContents:BCAReagentA,1000mL(inProductNo.23225)or500mL(inProductNo.23227),containingsodiumcarbonate,sodiumbicarbonate,bicinchoninicacidandsodiumtartratein0.1MsodiumhydroxideBCAReagentB,25mL,containing4%cupricsulfateAlbuminStandardAmpules,2mg/mL,10×1mLampules,containingbovineserumalbumin(BSA)at2mg/mLin0.9%salineand0.05%sodiumazideStorage:Uponreceiptstoreatroomtemperature.Productshippedatambienttemperature.Note:IfeitherReagentAorReagentBprecipitatesuponshippingincoldweatherorduringlong-termstorage,dissolveprecipitatesbygentlywarmingandstirringsolution.Discardanykitreagentthatshowsdiscolorationorevidenceofmicrobialcontamination.TableofContentsIntroduction.................................................................................................................................................................................1PreparationofStandardsandWorkingReagent(requiredforbothassayprocedures)................................................................2TestTubeProcedure(SampletoWRratio=1:20).....................................................................................................................3MicroplateProcedure(SampletoWRratio=1:8)......................................................................................................................3Troubleshooting...........................................................................................................................................................................4RelatedThermoScientificProducts............................................................................................................................................5AdditionalInformation................................................................................................................................................................5References...................................................................................................................................................................................6IntroductionTheThermoScientificPierceBCAProteinAssayisadetergent-compatibleformulationbasedonbicinchoninicacid(BCA)forthecolorimetricdetectionandquantitationoftotalprotein.Thismethodcombinesthewell-knownreductionofCu+2toCu+1byproteininanalkalinemedium(thebiuretreaction)withthehighlysensitiveandselectivecolorimetricdetectionofthecuprouscation(Cu+1)usingauniquereagentcontainingbicinchoninicacid.1Thepurple-coloredreactionproductofthisassayisformedbythechelationoftwomoleculesofBCAwithonecuprousion.Thiswater-solublecomplexexhibitsastrongabsorbanceat562nmthatisnearlylinearwithincreasingproteinconcentrationsoverabroadworkingrange(20-2000µg/mL).TheBCAmethodisnotatrueend-pointmethod;thatis,thefinalcolorcontinuestodevelop.However,followingincubation,therateofcontinuedcolordevelopmentissufficientlyslowtoallowlargenumbersofsamplestobeassayedtogether.Themacromolecularstructureofprotein,thenumberofpeptidebondsandthepresenceoffourparticularaminoacids(cysteine,cystine,tryptophanandtyrosine)arereportedtoberesponsibleforcolorformationwithBCA.2Studieswithdi-,tri-andtetrapeptidessuggestthattheextentofcolorformationcausedbymorethanthemeresumofindividualcolor-producingfunctionalgroups.2Accordingly,proteinconcentrationsgenerallyaredeterminedandreportedwithreferencetostandardsofacommonproteinsuchasbovineserumalbumin(BSA).Aseriesofdilutionsofknownconcentrationarepreparedfromtheproteinandassayedalongsidetheunknown(s)beforetheconcentrationofeachunknownisdeterminedbasedonthestandardcurve.Ifprecisequantitationofanunknownproteinisrequired,itisadvisabletoselectaprotein1296.72322523227Pierce®BCAProteinAssayKitPierceBiotechnologyPOBox117(815)968-0747(815)968-7316fax2standardthatissimilarinqualitytotheunknown;forexample,abovinegammaglobulin(BGG)standard(seeRelatedThermoScientificProducts)maybeusedwhenassayingimmunoglobulinsamples.Twoassayproceduresarepresented.Ofthese,theTestTubeProcedurerequiresalargervolume(0.1mL)ofproteinsample;however,becauseitusesasampletoworkingreagentratioof1:20(v/v),theeffectofinterferingsubstancesisminimized.TheMicroplateProcedureaffordsthesamplehandlingeaseofamicroplateandrequiresasmallervolume(10-25µL)ofproteinsample;however,becausethesampletoworkingreagentratiois1:8(v/v),itofferslessflexibilityinovercominginterferingsubstanceconcentrationsandobtaininglowlevelsofdetection.PreparationofStandardsandWorkingReagent(requiredforbothassayprocedures)A.PreparationofDilutedAlbumin(BSA)StandardsUseTable1asaguidetoprepareasetofproteinstandards.DilutethecontentsofoneAlbuminStandard(BSA)ampuleintoseveralcleanvials,preferablyusingthesamediluentasthesample(s).Ea