ANALYTICALBIOCHEMISTRYARTICLENO.

247,185–192(1997)

AB972061

High-Ef ciencyBlottingofProteinsofDiverseSizesFollowingSodiumDodecylSulfate–PolyacrylamideGelElectrophoresis

MarkW.BoltandPaulA.Mahoney1

DivisionofBiologicalSciences,UniversityofMissouri,Columbia,Missouri65211

ReceivedSeptember11,1996

High-molecular-weightproteinsoftenblotontoni-trocellulosemembranespoorlyfollowingsodiumdo-decylsulfate–polyacrylamidegelelectrophoresis,re-sultinginlowlevelsofdetectiononimmunoblots,andhencedif cultyinanalyzingrareproteins.Moreover,optimizingconditionsforthetransferofhigh-molecu-lar-weightproteinstonitrocellulosefrequentlyresultsintheinef http://ingradiolabeledproteinstandardsandphos-phorimagingtechnology,wehavequantitatedtheef- cacyofmanydifferentproteingelelectrophoresisandblottingprotocols.Herewereportnovelgelandblottingconditionswhichsigni cantlyimprovethetransferandretentionofhigh-molecular-weightpro-teins,withoutsacri cingtheef http://ingthisnewlydescribedprocedure,wehavedetectedarare500-kDaproteininimmunoblotswhichwaspreviouslynotdetectablewithanyofthecommonlyusedblottingprocedures.Sincetheimprovedconditionsofferincreasedsensi-tivityacrossaspectrumofproteinsizes,theyshouldbewidelyapplicable. 1997AcademicPress

Theelectrophoretictransferofproteinsfrompoly-acrylamidegelstonitrocellulosesheets,initiallyde-scribedbyTowbin(1),hasenjoyedwidespreaduseasavaluabletoolinthe eldofproteinresearch.Oneapplicationofparticularimporthasbeenthesubse-quentemploymentofantibodyprobesdirectedtowardthenitrocellulose-boundproteins.Variouslytermedimmunoblotting,proteinblotting,or‘‘Western’’blotting

Towhomcorrespondenceshouldbeaddressed.Fax:573-882-0123.E-mail:mahoney@biosci.mbp.missouri.edu.

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Copyright 1997byAcademicPress

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(2),themethodologyofthisapplicationhaschangedlittlesinceitwasoriginallyintroducedforusewithurea-orSDS2-containingpolyacrylamidegels(1).Nu-merousattemptstoimprovetheoriginalprotocolhavefocusedonincreasingtheamountofproteintrans-ferredtoandretainedonthenitrocellulosesheet,par-ticularlywhendealingwitheitherverysmallorverylargeproteins.Theobservationthatsmallproteinstendtomovethroughthenitrocellulose,withonlyafractionofthetotalamountactuallybinding,ledtoananalysisshowingthatnitrocellulosewithsmallerporesizeswasmoreeffectiveinretainingsmallproteins(3).Morerecently,Tooetal.describetheuseofgelatin-coatednitrocelluloseforthequantitativeretentionofsmallproteins(4).Conversely,investigatorsanalyzinglargeproteinshaveoftensoughttoincreasetheef -ciencyoftransferbyenhancingthedegreeofproteinmigrationoutofthegelduringthetransfer,usingtech-niquesrangingfromdisruptionofthegelmatrixtopartialproteolyticdigestionoftheproteinspriortotransfer(5–7).Theseexampleshighlighttwomajorfactorsin uencingtheef ciencyofproteinblotting:theelutionef ciencyofaproteinoutofagivengelmatrixandtheef ciencyofbindingbythemembrane.Im-provementsinoneareaoftenappeartobegainedatsomecostintheother;methanolhasbeenreportedtoincreasethebindingcapacityofnitrocellulose,perhapsthroughhydrophobiceffects(8),butitmaydecreasetheelutionef ciencyoflargeproteinsbypartially‘‘ xing’’theminthegel(forreview,see9).

Recently,oureffortstostudyalarge(ca.500kDa)membraneproteinwerestymiedbyourinabilitytode-tecttheproteinonimmunoblots.Suspectingthatthisre ectedapoorelutionef ciencyfromthegel,weem-barkedonasystematicstudyofsomeoftheparameters

Abbreviationsused:TEMED,N,N,N ,N -tetramethylenediamine;SDS,sodiumdodecylsulfate.

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commonlyemployedduringdenaturingSDS–http://ingradiolabeledproteinsandphosphorimaginganalysis,wewereabletoquantitatetransferef cienc-iesundermanydifferentconditions,includingthosecommonlyusedforelectrophoresisonaLaemmli-typegel(10)followedbyproteinblotting(11,12).Ourre-sultsshowthatthemaindeterminantofproteinblot-tingef ciencyrestsnotintheabilityofproteinstoexitaLaemmli-typegelduringblotting,butratherintheef ciencyofproteinbindingtothenitrocelluloseblot-tingmembrane.Byalteringthecompositionofthebufferusedduringtheblottingstep,wewereabletoincreasetheretentiononnitrocelluloseofmostproteinstested.Mostimportantly,whenthismodi edblottingbufferwasusedincombinationwiththegelsystemdescribedbyFairbanksetal.(13),wefoundasigni cantincreaseintheoverallblottingef ciencyofproteins,overawiderangeofproteinsizes.Forthe200-kDamyosinheavychain,theamountofproteintransferredwasalmostfourfoldmorethanthatobtainedusingmoreconventionalconditions.Moreover,theuseofthisnewcombinationofgelsystemandmodi edbufferal-lowedustodetectahigh-molecular-weightproteininthe400-to500-kDarangethathadnotbeendetectedpreviouslyusingawidevarietyofexistinggelprepara-tionandblottingprocedures.Thus,webelievethattheconditionsdescribedhereforSDS–polyacrylamidegelelectrophoresis,followedbyblottingtoanitrocellulosemembraneresultinthehighlyef cienttransferofpro-teinfromthegeltothemembrane,arewidelyapplica-bletoproteinsofdiversesizesandareparticularlyusefulfordetectinglow-abundance,high-molecular-weightproteins.

MATERIALSANDMETHODS

Genie(IdeaScienti c,Minneapolis,MN)wettransferblottingapparatuswith10115-cmplateelectrodes.PolyacrylamideGelElectrophoresis

Gelpreparation.Analyticalgels(8018011mm)consistedofeithera5to20%(w/v)gradientresolvinggelcoupledtoa4%(w/v)stackinggel,preparedasde-scribedbyLaemmli(10),or3.3to15%(w/v)gradientgelsusingbuffersdescribedbyFairbanksetal.(13).Sincewewereprimarilyinterestedinhigh-molecular-weightproteins,wecastgelsusinga50:1ratioofacryl-amide:bisacrylamideinallexperiments.Allgelswerestoredatroomtemperatureovernighttoensurecom-pleteandconsistentpolymerization.

Laemmligels.Theresolvingportionofthesegradi-entgelsconsistedof5%(w/v)acrylamide,375mMTris–HCl(pH8.8),0.1%(w/v)SDS,5%(v/v)glycerol,0.15%(w/v)ammoniumpersulfate,0.2%(v/v)TEMEDastheuppergradientmixtureand20%(w/v)acrylamide,375mMTris–HCl(pH8.8),0.1%(w/v)SDS,15%(v/v)g …… 此处隐藏：26309字，全部文档内容请下载后查看。喜欢就下载吧 ……