Protein-measurement-with-the-Folin-phenol-reagent

PROTEINMEASUREMENTWITHTHEFOLINPHENOLREAGENT*BYOLIVERH.LOWRY,NIRAJ.ROSEBROUGH,A.LEWISFARR,ANDROSEJ.RANDALL(FromtheDepartmentofPharmacology,WashingtonUniversitySchoolojMedicine,St.Louis,Missouri)(Receivedforpublication,May28,1951)Since1922whenWuproposedtheuseoftheFolinphenolreagentforthemeasurementofproteins(l),anumberofmodifiedanalyticalpro-ceduresut.ilizingthisreagenthavebeenreportedforthedeterminationofproteinsinserum(2-G),inantigen-antibodyprecipitates(7-9),andininsulin(10).Althoughthereagentwouldseemtoberecommendedbyitsgreatsen-sitivityandthesimplicityofprocedurepossiblewithitsuse,ithasnotfoundgreatfavorforgeneralbiochemicalpurposes.Inthebeliefthatthisreagent,nevertheless,hasconsiderablemeritforcertainapplication,butthatitspeculiaritiesandlimitationsneedtobeunderstoodforitsfullestexploitation,ithasbeenstudiedwithregardt.oeffectsofvariationsinpH,timeofreaction,andconcentrationofreact-ants,permissiblelevelsofreagentscommonlyusedinhandlingproteins,andinterferingsubst.ances.Proceduresaredescribedformeasuringpro-teininsolutionorafterprecipitationwit,hacidsorotheragents,andforthedeterminationofaslittleas0.2yofprotein.MethodReagents-ReagentA,2percentN&OXin0.10NNaOH.ReagentB,0.5percentCuS04.5Hz0in1percentsodiumorpotassiumtartrabe.ReagentC,alkalinecoppersolution.Mix50ml.ofReagentAwith1ml.ofReagentB.Discardafter1day.ReagentD,carbonate-coppersolution,isthesameasReagentCexceptforomissionofNaOH.Re-agentE,dilutedFolinreagent.TitrateFolin-Ciocalteuphenolreagent((II),EimerandAmend,FisherScientificCompany,NewYork)withNaOHt.oaphenolphthaleinend-point.OnthebasisofthistitrationdilutetheFolinreagent(about2-fold)tomakeit1Ninacid.WorkingstandardsmaybepreparedfromhumanserumdilutedIOO-tolOOO-fold(approximately700to70yperml.).Theseinturnmaybecheckedagainstastandardsolutionofcrystallinebovinealbumin(Armourand*SupportedinpartbyagrantfromtheAmericanCancerSocietyontherecom-mendationoftheCommitteeonGrowthoftheNationalResearchCouncil.265byguest,onJanuary10,2011);1yistheequivalentof0.97yofserumprotein(seebelow).Dilutesolutionsofbovinealbuminhavenotprovedsatisfactoryforworkingstandardsbecauseofamarkedtendencytoundergosurfacedenaturation.ProcedureforProteinsinSolutionorReadilySolubleinDiluteAlkali-(Directionsaregivenforafinalvolumeof1.1to1.3ml.,butanymultipleorfractionofthevolumesgivenmaybeemployedasdesired’.)Toasampleof5to100yofproteinin0.2ml.orlessina3to10ml.test-tube,1ml.ofReagentCisadded.Mixwellandallowtostandfor10minutesorlongeratroomtemperature.0.10ml.ofReagentEisaddedveryrapidlyandmixedwithinasecondort,wo(seebelow).After30minutesorlonger,thesampleisreadinacalorimeterorspectrophotome-ter.Fortherange5to25yofproteinperml.offinalvolume,itisdesirabletomakereadingsatornearX=750rnp,theabsorptionpeak.Forstrongersolutions,thereadingsmaybekeptinaworkablerangebyreadingnearX=500m/l(Fig.2).Calculatefromastandardcurve,and,ifnecessary,makeappropriatecorrectionfordifferencesbetweenthecolorvalueoftheworkingstandardandtheparticularproteinsbeingmeasured(seebelow).Itisunnecessarytobringallthesamplesandstandardstothesamevolumebeforetheadditionofthealkalinecopperreagent,providedcor-rectionsaremadeforsmalldifferencesinfinalvolume.Thecriticalvol-umesarethoseofthealkalinecopperandFolinreagents.Iftheproteinispresentinanalreadyverydilutesolution(lessthan25yperml.),0.5ml.maybemixedwith0.5ml.ofanexactlydoublestrengthReagentCandotherwisetreatedasabove.InsolubleProteins,etc.-Manyproteinprecipitates,e.g.tungstatepre-cipitates,willdissolvereadilyinthealkalinecopperreagent.However,afterproteinshavebeenprecipitatedwithtrichloroaceticorperchloricacid,forexample,theywilldissolveratherpoorlyinthe0.1Nalkaliofthisreagent.Theybecomeevenhardertodissolveifsubsequentlyex-tract.edwithfatsolvents,andstillmoresoifdriedat100’.Itisnotpossibletocoverallcases,butthefollowingmaybehelpfulinmeasuringtheproteinofacidprecipitates.Iftheamountofproteinisnotgreat,sothatitisspreadratherthinly,itwillusuallydissolvein3hourorsoin1NNaOHatroomtemperature.Therefore,onemayadd,forexample,0.1ml.of1NNaOHto5to100yofprecipitatedprotein.1Forexample,withtheKlettcalorimeter,transfer25to500yofproteininnotover1ml.volumetoacalorimetertube.Addwaterifnecessarytomake1ml.Add5ml.ofReagentC,and,after10minutes,0.5ml.ofReagentE.Readingsaretakenafter30minuteswiththeNo.66filter.Ifthereadingsaretoohigh,substi-tutetheNo.54filterforsample,standards,andblanks.byguest,onJanuary10,2011(noNaOH)isadded,followedafter10minutesby0.1ml.ofdilutedFolinReagentEasusual.Withlargersamples,orverystubbornprecipitates,itmaybenecessarytoheatfor10minutesormoreat100”in1Nalkali.Althoughthismaylowerthereadings,theywillbereproducibleandcanbemeasuredwithsimilarlytreatedstandards.2Microanalysis-WithaBeckmanspectrophotometeradaptedto0.05ml.volume(12),aslittleas0.2yofproteinmaybemeasuredwithreason-ableprecision.Asidefromreducingthevolumesofsampleandreagents,theonlynecessarychangeisto