Bradford protein assay

Bradfordproteinassay

Considerationsforuse

TheBradfordassayisveryfastandusesaboutthesameamountofproteinastheLowryassay.Itisfairlyaccurateandsamplesthatareoutofrangecanberetestedwithinminutes.TheBradfordisrecommendedforgeneraluse,especiallyfordeterminingproteincontentofcellfractionsandassesingproteinconcentrationsforgelelectrophoresis.

Assaymaterialsincludingcolorreagent,proteinstandard,andinstructionbookletareavailablefromBio-RadCorporation.Themethoddescribedbelowisforthe"standardProcedure"withsensitivitytoabout20to200microgramsprotein.Simplyscaledownthevolumeforthe"microassayprocedure,"whichuses1mlcuvettes.Microtiterplateprotocolsarealsodescribedintheflyerthatcomeswiththekit.

Principle

TheassayisbasedontheobservationthattheabsorbancemaximumforanacidicsolutionofCoomassieBrilliantBlueG-250shiftsfrom465nmto595nmwhenbindingtoproteinoccurs.BothhydrophobicandionicinteractionsstABIlizetheanionicformofthedye,causingavisIBLecolorchange.Theassayisusefulsincetheextinctioncoefficientofadye-albumincomplexsolutionisconstantovera10-foldconcentrationrange.

Equipment

Inadditiontostandardliquidhandlingsuppliesavisiblelightspectrophotometerisneeded,withmaximumtransmissionintheregionof595nm,ontheborderofthevisiblespectrum(nospeciallamporfilterusuallyneeded).Glassorpolystyrene(cheap)cuvettesmaybeused,howeverthecolorreagentstainsboth.Disposablecuvettesarerecommended.

Procedure

Reagents

Bradfordreagent:Dissolve100mgCoomassieBrilliantBlueG-250in50ml95%ethanol,add100ml85%(w/v)phosphoricacid.Diluteto1literwhenthedyehascompletelydissolved,andfilterthroughWhatman#1paperjustbeforeuse.
(Optional)1MNaOH(tobeusedifsamplesarenotreADIlysolubleinthecolorreagent).

TheBradfordreagentshouldbealightbrownincolor.Filtrationmayhavetoberepeatedtoridthereagentofbluecomponents.TheBio-Radconcentrateisexpensive,butthelotsofdyeusedhaveapparentlybeenscreenedformaximumeffectiveness."Homemade"reagentworksquitewellbutisusuallynotassensitiveastheBio-Radproduct.

Assay

Warmupthespectrophotometer15min.beforeuse.
Dilutesampleswithbuffertoanestimatedconcentrationof20to200micrograms/ml
Preparestandardscontainingarangeof20to200microgramsprotein(albuminorgammaglobulinarerecommended)toastandardvolume(generally1mlorless).Seehowtoprepareanduseaproteinstandardcurveforsuggestionsastosettingupthestandards.
Prepareunknownstoestimatedamountsof20to200microgramsproteinpertube,samevolumeastheunknowns.
(optional)Add0.25ml1MNaOHtoeachsampleandvortex.
Add5mldyereagentandincubate5min.
Measuretheabsorbanceat590nm.

Analysis

Prepareastandardcurveofabsorbanceversusmicrogramsprotein(orviceversa),anddetermineamountsfromthecurve.Determineconcentrationsoforiginalsamplesfromtheamountprotein,volume/sample,anddilutionfactor,ifany.Ifyouareunfamiliarwithhowtoobtainaproteinconcentrationforadilutedsamplefromastandardcurve,seehowtoprepareanduseaproteinstandardcurve.

Comments

Thedyereagentreactsprimarilywitharginineresiduesandlesssowithhistidine,lysine,tyrosine,tryptophan,andphenylalanineresidues.Obviously,theassayislessaccurateforbasicoracidicproteins.TheBradfordassayisrathersensitivetobovineserumalbumin,moresothan"average"proteins,byaboutafactoroftwo.ImmunogloginG(IgG-gammaglobulin)isthepreferredproteinstandard.Theadditionof1MNaOHwassuggestedbyStoscheck(1990)toallowthesolubilizationofmembraneproteinsandreducetheprotein-to-proteinvariationincoloryield.

References

Bradford,MM.Arapidandsensitiveforthequantitationofmicrogramquantititesofproteinutilizingtheprincipleofprotein-dyebinding.AnalyticalBiochemistry72:248-254.1976.
Stoscheck,CM.QuantitationofProtein.MethodsinEnzymology182:50-69(1990).

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