Lowry – Protein Determination

Introduction

ThesensitivityoftheprocedureofLowryismoderatelyconstantfromproteintoprotein,andithasbeensowidelyusedthatLowryproteinestimationsareacompletelyacceptablealternativetoarigorousabsolutedeterminationinalmostallcircumstanceswhereproteinmixturesorcrudeextractsareinvolved.

ThemethodisbasedonboththeBiuretreaction,wherethepeptidebondsofproteinsreactwithcopperunderalkalineconditionsproducingCu+,whichreactswiththeFolinreagent,andtheFolin-Ciocalteaureaction,whichispoorlyunderstoodbutinessencephosphomolybdotungstateisreducedtoheteropolymolybdenumbluebythecopper-catalyzedoxidationofaromaticaminoacids.Thereactionsresultinastrongbluecolor,whichdependspartlyonthetyrosineandtryptophancontent.Themethodissensitivedowntoabout0.01mgofprotein/mL,andisbestusedonsolutionswithconcentrationsintherange0.01-1.0mg/mLofprotein.

Materials

1.Complex-formingreagent:PrepareimmediatelybeforeusebymixingthefollowingthreestocksolutionsA,B,andCintheproportion100:1:1(v:v:v),respectively.SolutionA:2%(w/v)Na₂CO₃indistilledwater.SolutionB:1%(w/v)CuSO₄·5H₂Oindistilledwater.SolutionC:2%(w/v)sodiumpotassiumtartrateindistilledwater.2.2NNaOH.3.Folinreagent(commerciallyavailable).Dilute1:2inH₂Obeforeuse.4.Standards:Useastocksolutionofstandardprotein(e.g.,bovineserumalbuminfractionV)containing4mg/mLproteinindistilledwaterstoredfrozenat-20°C.Preparestandardsbydilutingthestocksolutionwithdistilledwaterasfollows:

stocksolut.,ul01.252.56.2512.52562.5125250water,ul500499498494488475438375250Prot.conc.,ug/ml010205010020050010002000

Method

1.To0.2mLofsampleorstandardadd1mLoffreshlymixedcomplex-formingreagent.Letthesolutionstandatroomtemperaturefor10min

2.Add0.1mLofdilutedFolinreagent,usingavortexmixer,andletthemixturestandatroomtemperaturefor30-60min(donotexceed60min)

3.Readtheabsorbanceat750nmiftheproteinconcentrationwasbelow500ug/mLorat550nmiftheproteinconcentrationwasbetween100and2000ug/mL.

4.Plotastandardcurveofabsorbanceasafunctionofinitialproteinconcentrationanduseittodeterminetheunknownproteinconcentrations.

Notes

1.Ifthesampleisavailableasaprecipitate,thendissolvetheprecipitatein2NNaOH.

2.Petersonhasdescribedaprecipitationstepthatallowstheseparationoftheproteinsamplefrominterferingsubstancesandalsoconsequentlyconcentratestheproteinsample,allowingthedeterminationofproteinsindilutesolution.Peterson"sprecipitationstepisasfollows:a.Add0.1mLof0.15%deoxycholateto1.0mLofproteinsample.b.Vortex,andstandatroomtemperaturefor10min.c.Add0.1mLof72%TCA,vortex,andcentrifugeat10000rpmfor30min.d.Decantthesupernatantandthendissolvethepelletin2NNaOH.

3.ThereactionisverypH-dependent,anditisthereforeimportanttomaintainthepHbetween10and10.5.Takecare,therefore,whenanalyzingsamplesthatareinstrongbufferoutsidethisrange.

4.Theincubationperiodisnotcriticalandcanvaryfrom10mintoseveralhourswithoutaffectingthefinalabsorbance.

5.TheVortexstepiscriticalforobtainingreproducibleresults.TheFolinreagentisonlyreactiveforashorttimeunderthesealkalineconditions,beingunstableinalkali,andgreatcareshouldthereforebetakentoensurethoroughmixing.

6.Theassayisnotlinearathigherconcentrations.Ensure,therefore,thatyouareanalyzingyoursampleonthelinearportionofthecalibrationcurve.

7.Asetofstandardsisneededwitheachgroupofassays,preferablyinduplicate.Duplicateortriplicateunknownsarerecommended.

8.OnedisadvantageoftheLowrymethodisthefactthatarangeofsubstancesinterferewiththisassay,includingbuffers,drugs,nucleicacids,andsugars.Inmanycases,theeffectsoftheseagentscanbeminimizedbydilutingthemout,assumingthattheproteinconcentrationissufficientlyhightostillbedetectedafterdilution.Wheninterferingcompoundsareinvolved,itis,ofcourse,importanttorunanappropriateblank.Interferencecausedbydetergents,sucrose,andEDTAcanbeeliminatedbytheadditionofSDS.ThebestalternativeinthiscaseistodoLowry-TCA(seeProteinPrecipitationProtocols)orPeterson.

9.Modificationstothisbasicassayhavebeenreportedthatincreasethesensitivityofthereaction.IftheFolinreagentisaddedintwoportions,vortexingbetweeneachaddition,a20%increaseinsensitivityisachieved.Theadditionofdithiothreitol3minaftertheadditionoftheFolinreagentincreasesthesensitivityby50%.

10.Theamountofcolorproducedinthisassaybyanygivenprotein(ormixtureofproteins)isdependentontheaminoacidcompositionoftheprotein(s)(seeIntroduction).Therefore,twodifferentproteins,eachforexampleatconcentrationsof1mg/mL,cangivedifferentcoloryieldsinthisassay.Itmustbeappreciated,therefore,thatusingBSA(oranyotherproteinforthatmatter)asastandardonlygivesanapproximatemeasureoftheproteinconcentration.Theonlytimewhenthismethodgivesanabsolutevalueforproteinconcentrationiswhentheproteinbeinganalyzedisalsousedtoconstructthestandardcurve.Themostaccuratewaytodeterminetheconcentrationofanyproteinsolutionisaminoacidanalysis.

InterferingReagentsforLowry

Manydetergents,Urea,GuanidineHCl,highsucrose,Ammoniumsulphate,>0.1MTrisHCl,>1MNaAcetateorNaPhosphate,EDTA,reducingagents,etc.