ampule

INSTRUCTIONS

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NumberDescription

23225PierceBCAProteinAssayKit,sufficientreagentsfor500test-tubeor5000microplateassays

23227PierceBCAProteinAssayKit,sufficientreagentsfor250test-tubeor2500microplateassays

KitContents:

BCAReagentA,1000mL(inProductNo.23225)or500mL(inProductNo.23227),containing

sodiumcarbonate,sodiumbicarbonate,bicinchoninicacidandsodiumtartratein0.1Msodium

hydroxide

BCAReagentB,25mL,containing4%cupricsulfate

AlbuminStandardAmpules,2mg/mL,10×1mLampules,containingbovinerumalbumin(BSA)

at2mg/mLin0.9%salineand0.05%sodiumazide

Storage:tshippedatambienttemperature.

Note:IfeitherReagentAorReagentBprecipitatesuponshippingincoldweatherorduringlong-term

storage,danykitreagentthat

showsdiscolorationorevidenceofmicrobialcontamination.

TableofContents

Introduction.................................................................................................................................................................................1

PreparationofStandardsandWorkingReagent(requiredforbothassayprocedures)................................................................2

TestTubeProcedure(SampletoWRratio=1:20).....................................................................................................................3

MicroplateProcedure(SampletoWRratio=1:8)......................................................................................................................3

Troubleshooting...........................................................................................................................................................................4

RelatedThermoScientificProducts............................................................................................................................................5

AdditionalInformation................................................................................................................................................................5

References...................................................................................................................................................................................6

Introduction

TheThermoScientificPierceBCAProteinAssayisadetergent-compatibleformulationbadonbicinchoninicacid(BCA)for

thodcombinesthewell-knownreductionofCu+2toCu+1by

proteininanalkalinemedium(thebiuretreaction)withthehighlynsitiveandlectivecolorimetricdetectionofthecuprous

cation(Cu+1)usingauniquereagentcontainingbicinchoninicacid.1Thepurple-coloredreactionproductofthisassayisformed

ter-solublecomplexexhibitsastrongabsorbanceat

562nmthatisnearlylinearwithincreasingproteinconcentrationsoverabroadworkingrange(20-2000µg/mL).TheBCA

methodisnotatrueend-pointmethod;thatis,r,followingincubation,therateof

continuedcolordevelopmentissufficientlyslowtoallowlargenumbersofsamplestobeassayedtogether.

Themacromolecularstructureofprotein,thenumberofpeptidebondsandtheprenceoffourparticularaminoacids

(cysteine,cystine,tryptophanandtyrosine)arereportedtoberesponsibleforcolorformationwithBCA.2Studieswithdi-,

tri-andtetrapeptidessuggestthattheextentofcolorformationcaudbymorethanthemeresumofindividualcolor-

producingfunctionalgroups.2Accordingly,proteinconcentrationsgenerallyaredeterminedandreportedwithreferenceto

standardsofacommonproteinsuchasbovinerumalbumin(BSA).Ariesofdilutionsofknownconcentrationare

preparedfromtheproteinandassayedalongsidetheunknown(s)beforetheconcentrationofeachunknownisdetermined

iquantitationofanunknownproteinisrequired,itisadvisabletolectaprotein

1296.72322523227

Pierce®BCAProteinAssayKit

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2

standardthatissimilarinqualitytotheunknown;forexample,abovinegammaglobulin(BGG)standard(eRelated

ThermoScientificProducts)maybeudwhenassayingimmunoglobulinsamples.

e,theTestTubeProcedurerequiresalargervolume(0.1mL)ofproteinsample;

however,becauitusasampletoworkingreagentratioof1:20(v/v),theeffectofinterferingsubstancesisminimized.

TheMicroplateProcedureaffordsthesamplehandlingeaofamicroplateandrequiresasmallervolume(10-25µL)of

proteinsample;however,becauthesampletoworkingreagentratiois1:8(v/v),itofferslessflexibilityinovercoming

interferingsubstanceconcentrationsandobtaininglowlevelsofdetection.

PreparationofStandardsandWorkingReagent(requiredforbothassayprocedures)

ationofDilutedAlbumin(BSA)Standards

thecontentsofoneAlbuminStandard(BSA)ampuleinto

veralcleanvials,preferablyusingthesamediluentasthesample(s).Each1mLampuleof2mg/mLAlbuminStandardis

sufficienttoprepillbesufficientvolume

forthreereplicationsofeachdilutedstandard.

ationofDilutedAlbumin(BSA)Standards

DilutionSchemeforStandardTestTubeProtocolandMicroplateProcedure(WorkingRange=20-2,000µg/mL)

Vial

VolumeofDiluent

(µL)

VolumeandSourceofBSA

(µL)

FinalBSAConcentration

(µg/mL)

A0300ofStock2000

B125375ofStock1500

C325325ofStock1000

D175175ofvialBdilution750

E325325ofvialCdilution500

F325325ofvialEdilution250

G325325ofvialFdilution125

H400100ofvialGdilution25

I40000=Blank

DilutionSchemeforEnhancedTestTubeProtocol(WorkingRange=5–250µg/mL)

Vial

VolumeofDiluent

(µL)

VolumeandSourceofBSA

(µL)

FinalBSAConcentration

(µg/mL)

A700100ofStock250

B400400ofvialAdilution125

C450300ofvialBdilution50

D400400ofvialCdilution25

E400100ofvialDdilution5

F40000=Blank

ationoftheBCAWorkingReagent(WR)

followingformulatodeterminethetotalvolumeofWRrequired:

(#standards+#unknowns)×(#replicates)×(volumeofWRpersample)=totalvolumeWRrequired

Example:forthestandardtest-tubeprocedurewith3unknownsand2replicatesofeachsample:

(9standards+3unknowns)×(2replicates)×(2mL)=48mLWRrequired

Note:2.0mLoftheWRisrequiredforeachsampleinthetest-tubeprocedure,whileonly200µlofWRreagentis

requiredforeachsampleinthemicroplateprocedure.

eWRbymixing50partsofBCAReagentAwith1partofBCAReagentB(50:1,ReagentA:B).Fortheabove

example,combine50mLofReagentAwith1mLofReagentB.

Note:WhenReagentBisfirstaddedtoReagentA,turbidityisobrvedthatquicklydisappearsuponmixingtoyielda

clear,sstablefor

veraldayswhenstoredinaclodcontaineratroomtemperature(RT).

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ProcedureSummary(Test-tubeProcedure,StandardProtocol)

Test-tubeProcedure(SampletoWRratio=1:20)

e0.1mLofeachstandardandunknownsamplereplicateintoanappropriatelylabeledtesttube.

2.0mLoftheWRtoeachtubeandmixwell.

ndincubatetubesatlectedtemperatureandtime:

•StandardProtocol:37°Cfor30minutes(workingrange=20-2000µg/mL)

•RTProtocol:RTfor2hours(workingrange=20-2000µg/mL)

•EnhancedProtocol:60°Cfor30minutes(workingrange=5-250µg/mL)

Notes:

•Increasingtheincubationtimeortemperatureincreasthenet562nmabsorbanceforeachtestanddecreasboth

theminimumdetectionlevelofthereagentandtheworkingrangeoftheprotocol.

•UawaterbathtoheattubesforeitherStandard(37°Cincubation)orEnhanced(60°Cincubation)

aforced-airincubatorcanintroducesignificanterrorincolordevelopmentbecauofunevenheattransfer.

ltubestoRT.

espectrophotometertto562nm,uently,measure

theabsorbanceofallthesampleswithin10minutes.

Note:BecautheBCAassaydoesnotreachatrueendpoint,colordevelopmentwillcontinueevenaftercoolingtoRT.

However,becautherateofcolordevelopmentislowatRT,nosignificanterrorwillbeintroducedifthe562nm

absorbancemeasurementsofalltubesaremadewithin10minutesofeachother.

cttheaverage562nmabsorbancemeasurementoftheBlankstandardreplicatesfromthe562nmabsorbance

measurementofallotherindividualstandardandunknownsamplereplicates.

eastandardcurvebyplotti

concentrationinµg/standardcurvetodeterminetheproteinconcentrationofeachunknownsample.

MicroplateProcedure(SampletoWRratio=1:8)

e25µLofeachstandardorunknownsamplereplicateintoamicroplatewell(workingrange=20-2000µg/mL).

Note:Ifsamplesizeislimited,10µLofeachunknownsampleandstandardcanbeud(sampletoWRratio=1:20).

However,theworkingrangeoftheassayinthiscawillbelimitedto125-2000µg/mL.

200µLoftheWRtoeachwellandmixplatethoroughlyonaplateshakerfor30conds.

lateandincubateat37°Cfor30minutes.

etheabsorbanceatornear562nmonaplatereader.

Notes:

•Wavelengthsfrom540-590nmhavebeenudsuccessfullywiththismethod.

•Becauplatereadersuashorterlightpathlengththancuvettespectrophotometers,theMicroplateProcedure

requiresagreatersampletoer

562nmmeasurementsaredesired,increatheincubationtimeto2hours.

•IncreasingtheincubationtimeorratioofsamplevolumetoWRincreasthenet562nmmeasurementforeachwell

andlowersbothtasall

standardsandunknownsaretreatedidentically,suchmodificationsmaybeuful.

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cttheaverage562nmabsorbancemeasurementoftheBlankstandardreplicatesfromthe562nmmeasurementsof

allotherindividualstandardandunknownsamplereplicates.

eastandardcurvebyplotti

concentrationinµg/standardcurvetodeterminetheproteinconcentrationofeachunknownsample.

Note:Ifusingcurve-fittingalgorithmsassociatedwithamicroplatereader,afour-parameter(quadratic)orbest-fitcurve

tingresultsbyhand,apoint-to-pointcurveispreferable

toalinearfittothestandardpoints.

Troubleshooting

ProblemPossibleCauSolution

NocolorinanytubesSamplecontainsacopperchelating

agent

Dialyze,desalt,ordilutesample

Increacopperconcentrationinworking

reagent(e.g.,u50:2,ReagentA:B)

Removeinterferingsubstancesfromsample

usingProductNo.23215

BlankabsorbanceisOK,but

standardsandsamplesshowless

colorthanexpected

Strongacidoralkalinebuffer,alters

workingreagentpH

Dialyze,desalt,ordilutesample

Colormeasuredatthewrong

wavelength

Measuretheabsorbanceat562nm

Colorofsamplesappearsdarker

thanexpected

ProteinconcentrationistoohighDilutesample

Samplecontainslipidsor

lipoproteins

Add2%SDStothesampletoeliminate

interferencefromlipids3

Removeinterferingsubstancesfromsample

usingProductNo.23215

Alltubes(includingblank)aredark

purple

BuffercontainsareducingagentDialyzeordilutesample

Removeinterferingsubstancesfromsample

usingProductNo.23215

Buffercontainsathiol

Buffercontainsbiogenicamines

(catecholamines)

Needtomeasurecolorata

differentwavelength

Spectrophotometerorplatereader

doesnothave562nmfilter

Colormaybemeasureatanywavelength

between540nmand590nm,althoughthe

slopeofstandardcurveandoverallassay

nsitivitywillbereduced

eringsubstances

CertainsubstancesareknowntointerferewiththeBCAassayincludingthowithreducingpotential,chelatingagents,and

etheyareknowntointerferewithproteinestimationatevenminuteconcentrations,avoidthe

followingsubstancesascomponentsofthesamplebuffer:

AscorbicAcidEGTAIronImpureSucro

CatecholaminesImpureGlycerolLipidsTryptophan

CreatinineHydrogenPeroxideMelibioTyrosine

CysteineHydrazidesPhenolRedUricAcid

OthersubstancesinterferetoalesrextentwithproteinestimationusingtheBCAassay,andthehaveonlyminor

(tolerable)mcompatibleconcentrationsformany

substancesintheStandardTestTubeProtocolarelistedinTable2(elastpageofInstructions).Substanceswere

compatibleattheindicatedconcentrationintheStandardTestTubeProtocoliftheerrorinproteinconcentrationestimation

caudbytheprenceofthesubstancewaslessthanorequalto10%.ThesubstancesweretestedusingWRprepared

-corrected562nmabsorbancemeasurements(fora1000µg/mLBSAstandard+

substance)werecomparedtothenet562nmmeasurementsofthesamestandardpreparedin0.9%m

compatibleconcentrationswillbelowerIntheMicroplateProcedurewherethesampletoWRratiois1:8(v/v).

Furthermore,itispossibletohaveasubstanceadditiveaffectsuchthateventhoughasinglecomponentisprentata

concentrationbelowitslistedcompatibility,asamplebuffercontainingacombinationofsubstancescouldinterferewiththe

assay.

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giesforeliminatingorminimizingtheeffectsofinterferingsubstances

TheeffectsofinterferingsubstancesinthePierceBCAProteinAssaymaybeeliminatedorovercomebyoneofveralmethods.

•Removetheinterferingsubstancebydialysisorgelfiltration.

•rategyiffectiveonlyifthestartingprotein

concentrationissufficienttoremainintheworkingrangeoftheassayupondilution.

•Precipitatetheproteinsinthesamplewithacetoneortrichloroaceticacid(TCA).Theliquidcontainingthesubstancethat

interferedisdiscardedandtheproteinpelletiasilysolubilizedinultrapurewaterordirectlyinthealkalineBCAWR.4

atively,ProductNo.23215maybeud(e

RelatedPierceProducts).

•IncreatheamountofcopperintheWR(prepareWRas50:2or50:3,ReagentA:B),whichmayeliminateinterference

bycopper-chelatingagents.

Note:Forgreatestaccuracy,theproteinstandardsmustbetreatedidenticallytothesample(s).

RelatedThermoScientificProducts

15041Pierce96-WellPlates,100/pkg.

15075ReagentRervoirs,200/pkg.

15036SealingTapefor96-WellPlates,100/pkg.

23209AlbuminStandardAmpules,2mg/mL,10×1mLampules,containingbovinerumalbumin(BSA)

23208Pre-DilutedProteinAssayStandards:BovineSerumAlbumin(BSA)Set,7×3.5mL

23212BovineGammaGlobulinStandard,2mg/mL,10×1mLampules

23213Pre-DilutedProteinAssayStandards,(BGG)Set,7×3.5mLaliquots

23235PierceMicroBCAProteinAssayKit,workingrangeof0.5-20µg/mL

23236CoomassiePlus(Bradford)AssayKit,workingrangeof1-1500µg/mL

23215Compat-Able™ProteinAssayPreparationReagentSet

23250PierceBCAProteinAssayKit−ReducingAgentCompatible

AdditionalInformation

visitourwebsiteforadditionalinformationincludingthefollowingitems:

•FrequentlyAskedQuestions

•TechTipprotocol:EliminateinterferingsubstancesfromsamplesforBCAProteinAssay

ativeTotalProteinAssayReagents

Ifinterferencebyareducingsubstanceormetal-chelatingsubstancecontainedinthesamplecannotbeovercome,trythe

ThermoScientificCoomassiePlus(Bradford)AssayKit(ProductNo.23236),whichislessnsitivetosuchsubstances.

ngandRe-usingGlassware

sswaremustbecleanedandgivenathoroughfinalrinwithultrapurewater.

characteristicsfordifferentproteins

Eachofthecommonlyudtotalproteinassaymethodxhibitssomedegreeofvaryingrespontowarddifferentproteins.

Thedifferencesrelatetoaminoacidquence,pI,structureandtheprenceofcertainsidechainsorprostheticgroupsthat

candramaticallyaltertheprotein’oteinassaymethodsuBSAorimmunoglobulin(IgG)asthe

standardagainstwhichtheconcentrationofproteininthesampleisdetermined(Figure1).However,ifgreataccuracyis

required,preparethestandardcurvefromapuresampleofthetargetprotein.

Tyteinsweretestedat1000µg/mLusingthe30-

minute/37°ragenetcolorresponforBSAwasnormalizedto1.00andtheaveragenetcolor

responoftheotherproteinsixpresdasaratiototheresponofBSA.

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Figure1:TypicalcolorresponcurvesforBSAandBGGusing

theStandardTestTubeProtocol(37°C/30-minuteincubation).

ance

ratios(562nm)forproteinsrelativetoBSAusing

theStandardTestTubeProtocol.

Ratio=(Avg“test”netAbs.)/(Abs.)

ProteinTestedRatio

Albumin,bovinerum1.00

Aldola,rabbitmuscle0.85

α-Chymotrypsinogen,bovine

1.14

CytochromeC,horheart0.83

Gammaglobulin,bovine1.11

IgG,bovine1.21

IgG,human1.09

IgG,mou1.18

IgG,rabbit1.12

IgG,sheep1.17

Insulin,bovinepancreas1.08

Myoglobin,horheart0.74

Ovalbumin0.93

Transferrin,human0.89

1.02

StandardDeviation0.15

CoefficientofVariation14.7%

CitedReferences

,P.K.,etal.(1985).m.150:76-85.

lman,K.,etal.(1988).Investigationofthebicinchoninicacidproteinassay:

Biochem.175:231-7.

r,estil,D.(1986).Interfm.159:138-42.

,R.,etal.(1989).Proteinmeasurementusingbicinchoninicacid:m.180:136-9.

ProductReferences

Adilakshami,ne,R.O.(2002).RibosomalproteinS25mRNApartnerswithMTF-1andLatoprovideap53-mediatedmechanismforsurvivalor

.277:4147-51.

Fischer,T.,etal.(1999).Clathrin-coatedve.96:6722-7.

Prozialeck,W.C.,etal.(2002).ChlamydiatrachomatisdisruptsN-cadherin-dependentcell-celljunctionsandquesterβ-catenininhumancervical

ionandImmunity70:2605-13.

Roberts,K.P.,etal.(2002).Acomparativeanalysisofexpressionandprocessingoftheratepididymalfluidandsperm-boundformsofproteinsDandE.

BiologyofReproduction67:525-33.

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iblesubstanceconcentrationsintheBCAProteinAssay(etextfordetails).§

Substance

Compatible

Concentration

Salts/Buffers

ACES,pH7.825mM

Ammoniumsulfate1.5M

Asparagine1mM

Bicine,pH8.420mM

Bis-Tris,pH6.533mM

Borate(50mM),pH8.5(#28384)undiluted

B-PER®Reagent(#78248)undiluted

CalciumchlorideinTBS,pH7.210mM

Na-Carbonate/Na-Bicarbonate(0.2M),pH9.4

(#28382)

undiluted

Cesiumbicarbonate100mM

CHES,pH9.0100mM

Na-Citrate(0.6M),Na-Carbonate(0.1M),pH9.0

(#28388)

1:8dilution*

Na-Citrate(0.6M),MOPS(0.1M),pH7.5(#28386)1:8dilution*

CobaltchlorideinTBS,pH7.20.8mM

EPPS,pH8.0100mM

FerricchlorideinTBS,pH7.210mM

Glycine•HCl,pH2.8100mM

Guanidine•HCl4M

HEPES,pH7.5100mM

Imidazole,pH7.050mM

MES,pH6.1100mM

MES(0.1M),NaCl(0.9%),pH4.7(#28390)undiluted

MOPS,pH7.2100mM

ModifiedDulbecco’sPBS,pH7.4(#28374)undiluted

NickelchlorideinTBS,pH7.210mM

PBS;Phosphate(0.1M),NaCl(0.15M),

pH7.2(#28372)

undiluted

PIPES,pH6.8100mM

RIPAlysisbuffer;50mMTris,150mMNaCl,

0.5%DOC,1%NP-40,0.1%SDS,pH8.0

undiluted

Sodiumacetate,pH4.8200mM

Sodiumazide0.2%

Sodiumbicarbonate100mM

Sodiumchloride1M

Sodiumcitrate,pH4.8orpH6.4200mM

Sodiumphosphate100mM

Tricine,pH8.025mM

Triethanolamine,pH7.825mM

Tris250mM

TBS;Tris(25mM),NaCl(0.15M),pH7.6(#28376)undiluted

Tris(25mM),Glycine(192mM),pH8.0(#28380)1:3dilution*

Substance

Compatible

Concentration

Detergents**

Brij®-355.0%

Brij-56,Brij-581.0%

CHAPS,CHAPSO5.0%

Deoxycholicacid5.0%

Octylβ-glucoside

5.0%

NonidetP-40(NP-40)5.0%

Octylβ-thioglucopyranoside

5.0%

SDS5.0%

Span®201.0%

Triton®X-1005.0%

TritonX-114,X-305,X-4051.0%

Tween®-20,Tween-60,Tween-805.0%

Zwittergent®3-141.0%

Chelatingagents

EDTA10mM

EGTA--------

Sodiumcitrate200mM

Reducing&Thiol-ContainingAgents

N-acetylglucosamineinPBS,pH7.210mM

Ascorbicacid--------

Cysteine--------

Dithioerythritol(DTE)1mM

Dithiothreitol(DTT)1mM

Gluco10mM

Melibio--------

2-Mercaptoethanol0.01%

Potassiumthiocyanate3.0M

Thimerosal0.01%

ts&Solvents

Acetone10%

Acetonitrile10%

Aprotinin10mg/L

DMF,DMSO10%

DMSO10%

Ethanol10%

Glycerol(Fresh)10%

Hydrazides--------

Hydrides(Na2BH4orNaCNBH3)--------

HydrochloricAcid100mM

Leupeptin10mg/L

*Dilutedwithultrapurewater.

**Detergentsweretestedusinghigh-purityThremoScientificSurfact-AmpsProducts,whichhavelowperoxidecontent.

--Dashed-lineentryindicatesthatthematerialisincompatiblewiththeassay.

§Foramoreextensivelistofsubstances,downloadTechTip#68:

TechTipincludescompatiblesubstancesforallofourproteinassaysandenableasycomparisons.