Gradient-SDS-Polyacrylamide-Gel-Electrophoresis-of

PolyacrylamideGelElectrophoresisofProteins6969From:TheProteinProtocolsHandbook,2ndEditionEditedby:J.M.Walker©HumanaPressInc.,Totowa,NJ12GradientSDSPolyacrylamideGelElectrophoresisofProteinsJohnM.Walker1.IntroductionThepreparationoffixed-concentrationpolyacrylamidegelshasbeendescribedinChapters10and11.However,theuseofpolyacrylamidegelsthathaveagradientofincreasingacrylamideconcentration(andhencedecreasingporesize)cansometimeshaveadvantagesoverfixed-concentrationacrylamidegels.Duringelectrophoresisingradientgels,proteinsmigrateuntilthedecreasingporesizeimpedesfurtherprogress.Oncethe“porelimit”isreached,theproteinbandingpatterndoesnotchangeapprecia-blywithtime,althoughmigrationdoesnotceasecompletely(1).Therearetwomainadvantagesofgradientgelsoverlineargels.First,amuchgreaterrangeofproteinMrvaluescanbeseparatedthanonafixed-percentagegel.Inacomplexmixture,verylow-mol-wtproteinstravelfreelythroughthegeltobeginwith,andstarttoresolvewhentheyreachthesmallerporesizetowardthelowerpartofthegel.Muchlargerproteins,ontheotherhand,canstillenterthegelbutstarttoseparateimmediatelyowingtothesievingeffectofthegel.ThesecondadvantageofgradientgelsisthatproteinswithverysimilarMrvaluesmayberesolved,whichotherwisecannotresolveinfixedpercentagegels.Aseachproteinmovesthroughthegel,theporesizebecomesmalleruntiltheproteinreachesitsporesizelimit.Theporesizeinthegelisnowtoosmalltoallowpassageoftheprotein,andtheproteinsamplestacksupatthispointasasharpband.Asimilar-sizedprotein,butwithslightlylowerMr,willbeabletotravelalittlefurtherthroughthegelbeforereachingitsporesizelimit,atwhichpointitwillformasharpband.Thesetwoproteins,ofslightlydifferentMrvalues,thereforeseparateastwo,close,sharpbands.Theusuallimitsofgradientgelsare3–30%acrylamideinlinearorconcavegradients.Thechoiceofrangewillofcoursedependonthesizeofproteinsbeingfractionated.Thesystemdescribedhereisfora5–20%lineargradientusingSDSpolyacrylamidegelelectrophoresis.ThetheoryofSDSpolyacrylamidegelelectrophoresishasbeendecribedinChapter11.2.Materials1.Stockacrylamidesolution:30%acrylamide,0.8%bis-acrylamide.Dissolve75gofacryl-amideand2.0gofN,N'-methylenebis-acrylamideinabout150mLofwater.Filterandmakethevolumeto250mL.Storeat4°C.Thesolutionisstableformonths.70Walker2.Buffers:a.1.875MTris-HCl,pH8.8.b.0.6MTris-HCl,pH6.8.Storeat4°C.3.Ammoniumpersulfatesolution(10%[w/v]).Makefreshasrequired.4.SDSsolution(10%[w/v]).Stableatroomtemperature.Incoldconditions,theSDScancomeoutofsolution,butmayberedissolvedbywarming.5.N,N,N',N'-Tetramethylenediamine(TEMED).6.Gradientformingapparatus(seeFig.1).Reservoirswithdimensionsof2.5cmidand5.0cmheightaresuitable.Thetworeservoirsofthegradientformershouldbelinkedbyflexibletubingtoallowthemtobemovedindependently.Thisisnecessarysincealthoughequalvolumesareplacedineachreservoir,thesolutionsdifferintheirdensitiesandtherelativepositionsofAandBhavetobeadjustedtobalancethetwosolutionswhentheconnectingclampisopened(seeNote3).3.Method1.Preparethefollowingsolutions:SolutionA,mLSolutionB,mL1.875MTris-HCl,pH8.83.03.0Water9.30.6Stockacrylamide,30%2.510.010%SDS0.150.15Ammoniumpersulfate(10%)0.050.05Sucrose—2.2g(equivalentto1.2mLvolume)2.Degaseachsolutionundervacuumforabout30sandthen,whenyouarereadytoformthegradient,addTEMED(12μL)toeachsolution.3.OncetheTEMEDisaddedandmixedin,poursolutionsAandBintotheappropriatereservoirs(seeFig.1.)4.Withthestirrerstirring,fractionallyopentheconnectionbetweenAandBandadjusttherelativeheightsofAandBsuchthatthereisnoflowofliquidbetweenthetworeservoirs(easilyseenbecauseofthedifferenceindensities).Donotworryifthereissomemixingbetweenreservoirs—thisisinevitable.5.Whenthelevelsarebalanced,completelyopentheconnectionbetweenAandB,turnthepumpon,andfillthegelapparatusbyrunningthegelsolutiondownoneedgeofthegelslab.Surprisingly,verylittlemixingwithinthegradientoccursusingthismethod.Apumpspeedofabout5mL/minissuitable.Ifapumpisnotavailable,thegradientmayberunintothegelundergravity.6.Whenthelevelofthegelreachesabout3cmfromthetopofthegelslab,connectthepumptodistilledwater,reducepumpspeed,andoverlaythegelwith2–3mmofwater.7.Thegradientgelisnowlefttosetfor30min.Remembertorinseoutthegradientformerbeforetheremaininggelsolutionsetsinit.8.Whentheseparatinggelhasset,prepareastackinggelbymixingthefollowing:a.1.0mL0.6MTris-HCl,pH6.8;b.1.35mLStockacrylamide;c.7.5mLWater;d.0.1mL10%SDS;e.0.05mLAmmoniumpersulfate(10%).PolyacrylamideGelElectrophoresisofProteins719.Degasthismixtureundervacuumfor30sandthenaddTEMED(12μL).10.Pouroffthewateroverlayeringthegelandwashthegelsurfacewithabout2mLofstackinggelsolutionandthendiscardthissolution.11.Thegelslabisnowfilledtothetopoftheplateswithstackinggelsolutionandthewell-formingcombplacedinposition(seeChapter11).12.Whenthestackinggelhasset(~15min),carefullyremovethecomb.Thegelisnowreadyforrunning.TheconditionsofrunningandsamplepreparationareexactlyasdescribedforSDSgelelectrophoresisinChapter11.4.Notes1.ThetotalvolumeofliquidinreservoirsAandBshouldbechosensuchthatitapproxi-matestothevolumeavailablebetweenthegelplates.However,allowancemustbemad