BCA法测蛋白含量.doc

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1、THEBICINCHONINICACID(BCA)ASSAYFORDETERMINATIONOFTOTALPROTEINPrinciple原理Smithetal.(1985)introducedthebicinchoninicacid(BCA)proteinassayreagent.Inonesense,itisamodificationoftheLowryproteinassayreagent.ThemechanismofcolorformationwithproteinfortheBCApro

2、teinassayreagentissimilartothatoftheLowryreagent,butthereareseveralsignificantdifferences.TheBCAproteinassayreagentcombinesthereductionofCu2+toCu+byproteininanalkalinemedium(i.e.,thebiuretreaction)withthehighlysensitiveandselectivecolorimetricdetectio

3、nofthecuprouscation(Cu+)bybicinchoninicacid.Thepurple-coloredreactionproductofthismethodisformedbythechelationoftwomoleculesofBCAwithonecuprousion(Fig.A.3H.3).TheBCA/coppercomplexiswater-solubleandexhibitsastronglinearabsorbanceat562nmwithincreasingpr

4、oteinconcentrations.TheprimaryadvantageoftheBCAproteinassayreagentisthatmostsurfactants,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，

5、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。Materials试剂材料Proteinstandard:2mg/mLBSA20mgBSAin10mLof0.9%NaClcontaining0.05%(w/v)sodiumazide.Storeupto6monthsat4°C.20mg牛血清蛋白+10mL含有0.9%NaCl和0.05%(w/v)的叠氮钠。4°C放置6个月。SamplebufferorsolventProteinsample

6、BCAreagentA:1g4,4’-dicarboxy-2,2’-biquinoline,disodiumsalt(Na2BCA;1%w/vfinal)二喹啉甲酸二钠盐2gNa2CO3·H2O(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Dissolvealloftheabovechemicals

7、exceptthesodiumbicarbonateindeionizedwaterandadjustthefinalvolumeto100mL.AdjustthepHto11.25byaddingthesodiumbicarbonatealittleatatime.Storethisalkalinereagentinaplasticcontainer1to3weeksatroomtemperature,longerat4°C.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

8、，一次加一点，调pH到11.25。试剂储存于塑料瓶中常温1-3周，4°C可放置更久。OnlythedisodiumsaltofBCAissolubleatneutralpH;thefreeacidisnotreadilysoluble.只有二钠盐BCA在中性条件下可溶；其他形式溶解性不好。BCAreagentB:4gCuSO4·5H2O(4%w/vfinal)in100mLH2O.Storeupto6monthsatroomtemperature.4g五水硫酸铜+100mL去离子水