BCA法测蛋白含量

1、CurrentProtocolsinCellBiologyPublishedonlineSeptember2011inWileyOnlineLibraryTHEBICINCHONINICACID(BCA)ASSAYFORDETERMINATIONOFTOTALPROTEINPrincipl原理Smithetal.(1985)introducedthebicinchoninicacid(BCA)proteinassayreagent.Inonesense,itisamodificationoftheLowryproteinassayreagent.Themechanismofcolorforma

2、tionwithproteinfortheBCAproteinassayreagentissimilartothatoftheLowryreagent,butthereareseveralsignificantdifferences.TheBCAproteinassayreagentcombinesthereductionofCu2+toCu+byproteininanalkalinemedium(i.e.,thebiuretreaction)withthehighlysensitiveandselectivecolorimetricdetectionofthecuprouscation(Cu

3、+)bybicinchoninicacid.Thepurple-coloredreactionproductofthismethodisformedbythechelationoftwomoleculesofBCAwithonecuprousion(Fig.A.3H.3).TheBCA/coppercomplexiswater-solubleandexhibitsastronglinearabsorbanceat562nmwithincreasingproteinconcentrations.TheprimaryadvantageoftheBCAproteinassayreagentistha

4、tmostsurfactants,evenifpresentinthesampleatconcentrationsupto5%(v/v),arecompatiblewiththismethod.TableA.3H.2isabrieftroubleshootingguideforthistechnique.二喹啉甲酸(BCA)法是在福林酚法的基础上改善而来的，即在碱性条件下蛋白质将二价铜离子还原成一价铜离子，然后与BCA试剂反应生成紫色化合物，在562nm处检测。step1;protein-Cu&lstep2：Cu1+2BCAOH-COO-CQOBOACltcomplexFigureAu3H3T

5、h-enascticxischimaliqfortheBCAPiqteinA韦韦ay.TableA.3H2TroubieEhootingGuidatorBCAProteinPfobfcmicau&cSolutionNuwLorinanylubesBlankA讯isnumial,bulstandardsandsamplesshowIcsacxiliirthanexpectedCtJcrd!sijmnplFEjppeudarkerIh烈ncxpecLcdSimngaridmiilkMinebuffer,altersworkingreogentplICoIoTtYLa$urdal(Ihtwronga

6、vJenhAJlluhs(iDcliadinj;曲氐hloiiik)arcdarkpurplePrJleinconceJiiLrslianLu62ntnAdd2%(uVvjSDSioibesaniple10tdiiLiiiiLi比iiLErfiEreftLeInxihlipidsJ)iolyzcnrdiluteIhes-AmplePripicnrethr卩rnEeinwhhIjk/hh.miCi和记rKiI(TCA);i.inlJetixythuliLittDOCi.ditsolwpelleiinBCAworkingacdgenl1rccrt1hcSiunpltwithiflctoacetai

7、nidi1；forthiol钉Cokifmaybereadatanyuweiengchtvlwcen550mnMild57(5tinMaterials试剂材料Proteinstandard:2mg/mLBSA20mgBSAin10mLof0.9%NaClcontaining0.05%(w/v)sodiumazide.Storeupto6monthsat4C.20mg牛血清蛋白+10mL含有0.9%NaCl和0.05%(w/v)的叠氮钠。4C放置6个月。SamplebufferorsolventProteinsampleBCAreagentA:g4,4-dicarboxy-2,2-biquino

8、line,disodiumsalt(Na2BCA;1%w/vfinal)二喹啉甲酸二钠盐gNa2CO3H2O(2%w/vfinal)一水合碳酸钠160mgsodiumtartratedihydrate(0.16%w/vfinal)二水合酒石酸钠0.4gNaOH(0.4%w/vfinal)氢氧化钠0.95gNaHCO3(0.95%w/vfinal)碳酸氢钠Dissolvealloftheabovechemicalsexceptthesodiumbicarbonateindeionizedwaterandadjustthefinalvolumeto100mL.AdjustthepHto11.25b

9、yaddingthesodiumbicarbonatealittleatatime.Storethisalkalinereagentinaplasticcontainer1to3weeksatroomtemperature,longerat4C.除了碳酸氢钠，把其他试剂都加入100mL去离子水中，然后分次加碳酸氢钠，一次加一点，调pH到11.25。试剂储存于塑料瓶中常温1-3周，4C可放置更久。OnlythedisodiumsaltofBCAissolubleatneutralpH;thefreeacidisnotreadilysoluble.只有二钠盐BCA在中性条件下可溶；其他形式溶解性不

10、好。BCAreagentB:4gCuSOj5H2O(4%w/vfinal)in100mL巧0.Storeupto6monthsatroomtemperature.4g五水硫酸铜+100mL去离子水。室温可放置6个月。BCAworkingreagent:Mix100partsBCAreagentAwith2partsreagentB.将100份的BCA试剂A和2份的BCA试剂B混合起来。Procedures过程Prepareadilutionseriesof2mg/mLBSAinsamplebufferordiluenttocoverarangefrom125to2000yg/mL.稀释2mg/

11、mLBSA制备浓度范围1252000卩g/mL。Add100yLsample,dilutedstandard,orbuffer(blank)intoappropriatelylabeledtubes.在试管中加100yL的样品、标准溶液和空白。Add2mLBCAworkingreagentmixtoeachtube.Vorteximmediately.加2mLBCA工作液至每个试管，迅速混匀。Incubatesamplesandstandardsfor30minat37C,thencooltoroomtemperature.将样品和标准溶液及空白在37C保温30分钟，然后冷却至室温。Measu

12、rethecolorat562nm(A562)onaspectrophotometerzeroedwithdeionizedwater.以空白调零，在562nm处测吸光值。Plotastandardcurvebygraphingtheaveragenetorblank-correctedA562valuesforthestandardsversusproteinconcentrationinmicrogramspermilliliter.ExamplecolorresponsecurvesforBSAandBGGareshowninFig.A.3H.4.以校正过的吸光值A562做曲线，蛋白浓度单位为yg/mL。以BSA或BGG做标准曲线的例子见图A.3H.4。Determinetheproteinconcentrationofthesamplebyinterpolationfromtheplot(seeStrategicPlanning).根据标准曲线的公式进行蛋白质浓度的计算。MS1.0FigureAa3H_4GraphutthecolorresponsecurvedobtainedwithPiercesBCAProteinAssayReagentusingbavinss