Tricine–SDS-PAGE

16|VOL.1NO.1|2006|NATUREPROTOCOLSPROTOCOLTricine–SDS-PAGEHermannSchäggerMolekulareBioenergetik,ZentrumderBiologischenChemie,UniversitätsklinikumFrankfurt,Theodor-Stern-Kai7,Haus26,D-60590Frankfurt,Germany.CorrespondenceshouldbeaddressedtoH.S.([e-mail]).Publishedonline12May2006;doi:10.1038/nprot.2006.4Tricine–SDS-PAGEiscommonlyusedtoseparateproteinsinthemassrange1–100kDa.Itisthepreferredelectrophoreticsystemfortheresolutionofproteinssmallerthan30kDa.Theconcentrationsofacrylamideusedinthegelsarelowerthaninotherelectrophoreticsystems.Theselowerconcentrationsfacilitateelectroblotting,whichisparticularlycrucialforhydrophobicproteins.Tricine–SDS-PAGEisalsousedpreferentiallyfordoubledSDS-PAGE(dSDS-PAGE),aproteomictoolusedtoisolateextremelyhydrophobicproteinsformassspectrometricidentification,anditoffersadvantagesforresolutionoftheseconddimensionafterblue-nativePAGE(BN-PAGE)andclear-nativePAGE(CN-PAGE).HereIdescribeaprotocolforTricine–SDS-PAGE,whichincludesefficientmethodsforCoomassieblueorsilverstainingandelectroblotting,therebyincreasingtheversatilityoftheapproach.Thisprotocolcanbecompletedin1–2d.INTRODUCTIONGlycine–SDS-PAGE(alsoknownasLaemmli–SDS-PAGE)1andTricine–SDS-PAGE2,3,basedonglycine-TrisandTricine-Trisbuffersystems,respectively,arethecommonlyusedSDSelectro-phoretictechniquesforseparatingproteins.Theacrylamidegelsusedareoftencharacterizedbythetotalpercentageconcentration(%T)ofbothmonomers(acrylamideandthecrosslinkerbisacrylamide)andthepercentageconcentrationofthecross-linker(%C)relativetothetotalconcentration4.Forsimplicity,hereIabbreviate(forexample)10%T,3%Cgels,as10%gels.Together,Laemmli–SDS-PAGEandTricine–SDS-PAGEcovertheproteinmassrange1–500kDa.Answeringthefollowingquestionsshouldhelpyoutoidentifytheoptimalsolutionforaspecificsep-arationproblemandhelpyoutodecidewhetherthisprotocolissuitableforyourexperimentalpurpose.Doesyoursamplecontainproteinsofinterestthatare100kDaand20kDa?Insuchasituationyouwillprobablyneedtousebothelectrophoresissystems.UseTricine–SDS-PAGEpreferentiallyfortheoptimalseparationofproteins30kDa,andLaemmli–SDS-PAGEforproteins30kDa.AdirectcomparisonoftheresolutioncapacityofTricine–SDS-PAGEandLaemmli–SDS-PAGEinthelowmolecularmassrangeisshowninFigure1.ThedifferentseparationcharacteristicsofthetwotechniquesaredirectlyrelatedtothestronglydifferingpKvaluesofthefunctionalgroupsofglycineandTricinethatdefinetheelectrophoreticmobilitiesofthetrailingions(glycineandTricine)relativetotheelectrophoreticmobilitiesofproteins.Someofthetheoreticalbackgroundofthesemethodshasbeendiscussed2.UniformacrylamideTricine–SDSgelscovernarrowmassranges—forexample,10%gelscovertherange1–100kDa(Fig.2a)and16%gelscovertherange1–70kDa(Fig.2b)—andofferhighresolution,especiallyforthesmallproteinandpeptiderange.Doublingthecrosslinkerconcentrationbyusing16%T,6%Cgels(Fig.2c)andincluding6Mureainthesegels(Fig.2d)increasestheresolutionofsmallproteinsfurther.TheseuniformacrylamideTricine–SDSgelsarealmostexclusivelyusedtosepa-rateverysmallproteinsandpeptides.Uniformhigh-acrylamideLaemmligelscannotbeusedtoaccessthesmallproteinrange,becausethestackinglimitintheLaemmlisystemistoohigh,andsmallproteinsusuallyappearassmearingbandsnearthegelfront.Inalessconvenientway,however,thesmallproteinandpeptiderangecanbeaccessedbymakinguseofgradientgelsthatcontinuouslydestackproteinsaccordingtodecreasingmassduringelectrophoresis.Laemmli-typegradientgels—forexample,8–16%and10–27%acrylamidegelsfortheranges6–250kDaand2–200kDa,respectively—coverwiderangesofmassbest.Whichstainingtechniquecanbeusedtovisualizethepro-teinsseparatedingels?Proteinbandscontainingaminimumof~0.2µgofproteinaresuitableforCoomassiestaining.Thestainingintensitiescanbeusedtoestimatethemolarratiosoftheproteinsubunitsofmultiproteincomplexesexcepttheveryhydrophobicsubunitsthatstainverypoorly.IdescribeaCoomassiestainingprotocolthatusesnomethanolinthestaininganddestainingsolutions;therefore,moreCoomassiedyeiskeptprotein-bound,andthesensitivityofthisCoomassiestainingprotocolisrelativelyhigh.Hundred-foldsmalleramountsofproteincanbevisualizedbyusingoneofthenumeroussilverstainingprotocols5;however,notallsilverstainingprotocolsseemtoworkwithTricine–SDSgels.Ipresentamodifiedversionoftheoriginalprotocol6,whichiscompatiblewithmassspectrometricanalysesandoffersoptimalstainingofhighlyhydrophobicproteins7.Doesyoursamplecontainhydrophobicproteinsofinterestthatyouwouldliketotransferontoinertmembranesanddetectbywesternblotting?Ifso,thenyoushoulduseTricine–SDS-PAGEandchooselow-acrylamidegels.Thelowpercentageofacryl-amidefacilitatesproteinblottingtomembranesandproteinrecoveryfromgels.Thisfeatureisessentialforveryhydrophobicproteinsthatotherwisemightnotbetransferredatallbycom-monelectrotransferprotocols.Variouselectroblottingprotocolsworkwellforwater-solubleproteins8–11;however,thetransferofveryhydrophobicproteinsiscrucialwithlargehydrophobicpro-teinsandhigh-acrylamidegels.Theseproteinscanbeefficientlytransferredbythisprotocol,whichuseshigh-ionic-strengthbuf-fer,lowvoltageandcorrespondingl