描述
TheThermoScientificPierceBCAProteinAssayKitisatwo-component,high-precision,detergent-compatIBLeassayreagentsettomeasure(A562nm)totalproteinconcentrationcomparedtoaproteinstandard.
FeaturesoftheBCAProteinAssayKit:
• Colorimetric—estimatevisuallyormeasurewithastandardspectrophotometerorplatereader(562nm)
• Excellentuniformity—exhibitslessprotein-to-proteinvariationthandye-bindingmethods
• Compatible—unaffectedbytypicalconcentrationsofmostionicandnonionicdetergents
• Moderatelyfast—mucheasierandfourtimesfasterthantheclassicalLowrymethod
• Highlinearity—linearworkingrangeforBSAequals20to2000µg/mL
• Sensitive—detectdownto5µg/mLwiththeenhancedprotocol
Usedinmorelabsthananyotherdetergent-compatibleproteinassay,PierceBCAReagentsprovideaccuratedeterminationofproteinconcentrationwithmostsampletypesencounteredinproteinresearch.ThePierceBCAAssaycanbeusedtoassessyieldsinwholecelllysatesandaffinity-columnfractions,aswellastomonitorproteincontaminationinindustrialapplications.Comparedtomostdye-bindingmethods,theBCAAssayisaffectedmuchlessbyproteincompositionaldifferences,providinggreaterprotein-to-proteinuniformity.
Applications:
•Studyingprotein:proteininteractions
•Measuringcolumnfractionsafteraffinitychromatography
•Estimatingpercentrecoveryofmembraneproteinsfromcellextracts
•High-throughputscreeningoffusionproteins
HowtheBCAProteinAssayDetectsProtein:
TheBCAProteinAssaycombinesthewell-knownreductionofCu2+ toCu1+ byproteininanalkalinemediumwiththehighlysensitiveandselectivecolorimetricdetectionofthecuprouscation(Cu1+)bybicinchoninicacid.Thefirststepisthechelationofcopperwithproteininanalkalineenvironmenttoformalightbluecomplex.Inthisreaction,knownasthebiuretreaction,peptidescontainingthreeormoreaminoacidresiduesformacoloredchelatecomplexwithcupricionsinanalkalineenvironmentcontainingsodiumpotassiumtartrate.
Inthesecondstepofthecolordevelopmentreaction,bicinchoninicacid(BCA)reactswiththereduced(cuprous)cationthatwasformedinstepone.Theintensepurple-coloredreactionproductresultsfromthechelationoftwomoleculesofBCAwithonecuprousion.TheBCA/coppercomplexiswater-solubleandexhibitsastronglinearabsorbanceat562nmwithincreasingproteinconcentrations.TheBCAreagentisapproximately100timesmoresensitive(lowerlimitofdetection)thanthepalebluecolorofthefirstreaction.
ThereactionthatleadstoBCAcolorformationisstronglyinfluencedbyfouraminoacidresidues(cysteineorcystine,tyrosine,andtryptophan)intheaminoacidsequenceoftheprotein.However,unliketheCoomassiedye-bindingmethods,theuniversalpeptidebackbonealsocontributestocolorformation,helpingtominimizevariABIlitycausedbyproteincompositionaldifferences.
