BCA法测蛋白含量.docx

建筑

建筑

Current

CurrentProtocolsinCellBiologyPublishedonlineSeptember2011inWileyOnlineLibrary

THEBICINCHONINICACID(BCA)ASSAYFORDETERMINATIONOFTOTALPROTEIN

Principle原理

Smithetal.(1985)introducedthebicinchoninicacid(BCA)proteinassayreagent.Inonesense,itisamodificationoftheLowryproteinassayreagent.ThemechanismofcolorformationwithproteinfortheBCAproteinassayreagentissimilartothatoftheLowryreagent,butthereareseveralsignificantdifferences.TheBCAproteinassayreagentcombinesthereductionofCu 2+toCu+byproteininanalkalinemedium(i.e.,thebiuretreaction)withthehighlysensitiveandselectivecolorimetricdetectionofthecuprouscation(Cu+)bybicinchoninicacid.Thepurple-coloredreactionproductofthismethodisformedbythechelationoftwomoleculesofBCAwithonecuprousion(Fig.A.3H.3).TheBCA/coppercomplexiswater-solubleandexhibitsastronglinearabsorbanceat562nmwithincreasing protein concentrations. TheprimaryadvantageoftheBCAproteinassayreagentisthatmostsurfactants,evenifpresentinthesample atconcentrations up to 5% (v/v), are compatible with this method. Table A.3H.2is a brieftroubleshootingguideforthistechnique.

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。

Materials试剂材料

Proteinstandard:2mg/mLBSA

20mgBSAin10mLof0.9%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

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

Dissolvealloftheabovechemicalsexceptthesodiumbicarbonateindeionizedwaterandadjustthefinalvolumeto100mL.AdjustthepHto11.25byaddingth