Two dimensional peptide mapping
ThisspecficprotocolisthelatestincarnationofpeptidemappingproceduresthathavebeendevelopedhereintheTVL/MBVLoftheSalkInstituteoverthelast20years.WadeGibsondevelopedthefirstpeptidemappingprotocol.ThiswasimprovedbyKarenBeemonandTonyHunter.TamaraHurleyandKunxinLuoadaptedittoproteinstransferredtofilters. 1.Immunoprecipitatetheproteinandrunitonapreparativegel.Scaleupyourimmunoprecipitationsothatyouhaveatleast500cpm.Transfertheproteintonitrocellulose,ImmobilonorNylonaccordingtoKamps\"sWesternProtocol.TrypticdigestioncanbedonewitheitherNC,Nylon,orImmobilon.NitrocelluloseseemstogivemorereproducIBLeyieldsofpeptides. 2.Rinseblotswithwateraftertransfer.WrapthemembraneinSaranwraptokeepthemembranewetduringexposure.Trytokeepthemembranedampthroughouttheprocedure.Ifitdriesout,youcanrewetNCwithwater,orImmobilonwithmethanolandthenwater,butyourrecoverywillprobablygodown.Afterexposure,cutoutthemembranepieceofinterestandsoakitimmediatelyin0.5%PVP-360(Polyvinylpyrrolidone,MW360)in100mMaceticacidfor30min.at37°C.Ifyoudon\"tdothis,youryieldwillbepoor.Thismayresultfromtheproteaseadsorbingontothefilter.3.Aspiratetheliquid.WashthemembraneextensivelywithH2O(5X1ml).Thenwashwithfreshly-made0.05MNH4HCO3onceortwice.4.Digestion:Add150-200microliterfresh0.05MNH4HCO3(enoughtocoverthepieceofmembrane).Startthedigestionbyadding10microgramstrypsinorchymotrypsin(Stockis1milligram/mlin0.05MNH4HCO3storedat-70°C).Mixbypipettingupanddownandincubateat37°Cfor2hr.5.Vortexthesampleandaddanother10microgramsofenzyme.Incubatefor2hrat37°C.6.Add300microliters(ormore)H2Otoeachsampleandspininmicrofugefor5minute.Transferthesupetonewtubeorremovethemembrane.Dryonthespeedvac.Ittakesabout3-4hours. 10.ThelocationoftheorigindependsonthepHoftheelectrophoresisbuffer.PeptidesaregenerallypositivelychargedatpH1.9or4.72andtheoriginisthereforedisplacedtowardthepositiveelectrodewhenusingthesebuffers.Asdiagrammedabove,theoriginforpH1.9orpH4.72is5cmfromtheleft(positive)edgeand2.5cmupfromthebottom. ThechargeofphosphopeptidesatpH8.9islesspredictableandtheoriginforthesemapsisusuallyplacedinthemiddleoftheplate,2.5cmupfromthebottom. 11.ItisimportanttorunMarkerdyes.Thegreendyeisa1:1mixtureofXylenecyanolFFandepsilon-DNPlysineinpH4.72buffer.Itshouldbespottedinapositionequivalenttothatofthesample,butinaportionoftheplatethatwillnotcontainpeptides. 12.Afterelectrophoresis,airdrytheplate.Itisthoughtthatdryingintheovenwillcookthepeptidesirreversiblytothecellulose. 13.Thencarryoutchromatography.Heretooyoushouldusethegreenmarkerdye.Spotitofftotheside,thesamedistanceupfromthebottomasthesample. 14.ThenmarktheplatewithrADIoactiveink.Itisusefultoputmarksonthatcanbeusedtofigureoutwheretheoriginwasandwherethemarkerdyesran.Don\"tmarktheoriginitself.Usually,3-4daysexposurewithascreenandflashedfilmisnecessarytodetectsamplescontaining100-200cpmof32P. It\"ssensibletomake2Loftheelectrophoresisbuffers.Thevolumeofchromatographybufferisdictatedbythesizeofyourtanks.Youneedenoughtogiveadepthofapproximately0.5cminthebottomofthetamk pH4.72buffer. ThepHshouldbeadjustedto4.72usingeitheraceticacidorpyridine.NotethattherecipeinWade\"spaperiswrong. ThepHshouldbeadjustedto8.90witheitherNH4OHorCO2 Don\"tusethe98%formicacidanddon\"tadjustthepH. Ifyouwanttoreadorcitethepapersdescribingthistechnique,theappropriatereferencesare: Gibson,W.(1974)Polyomavirusproteins:adescriptionofthestructuralproteinsofthevirionbasedonpolyacrylamidegelelectrophoresisandpeptideanalysis.Virology.62(2):319-36. Beemon,K.,andHunter,T.(1978)CharacterizationofRoussarcomavirussrcgeneproductssynthesizedinvitro.JVirol.28(2):551-66.[abstract] Luo,K.,Hurley,T.R.,andSefton,B.M.(1990)Transferofproteinstomembranesfacilitatesbothcyanogenbromidecleavageandtwo-dimensionalproteolyticmapping.Oncogene5(6):921-3.[abstract]![](\"dxy_expriment/201812/or_dot.gif\")
Transferoftheproteintoafilterismuchsuperiortohomogenizationoftheprepgelandelution.Althoughsomepeoplehaveconcernaboutinefficienttransferoflargeproteinsandlossofhydrophobicpeptidesonthefilters,wehavehadgoodsuccesswithessentiallyeveryproteinwehavestudied.![](\"dxy_expriment/201812/cy_dot.gif\")
Asageneralprinciple,itisvaluabletoworkfastandkeepthefilterdamp.Itisgenerallythoughtthatthelongertheproteinremainsboundtothefilter,thelowertheyield.
7.Oxidation:Thephosphorylatedtrypticpeptidesofp56lckdonotcontainMetorCysandoxidationisthereforenotnecessary.Ifthepeptidesyou\"reexaminingcontainMetorCys,orifyoudon\"tknow,youshouldoxidizethemwithperformicacid.Tomakeperformicacid,mix1vol.fresh30%H2O2with9vol.98%-100%formicacid.Incubatethemixtureatroomtemp.for1hrtoallowperformicacidtoform.Coolonice.Thenewly-madeperformicacidcanbestoredoniceforseveralhoursbeforeuse.ResUSPendthedriedpeptidesin50microlitersperformicacidbyvortexingorpipettingupanddown.Incubateonicefor2hr.(1hrisprobablyenough.Justbeconsistent).8.Add1mlH2Otothetube.DryonSpeed-vac.
Countsamples9.Resuspendthesamplein10microlitersH2Oforan8.9map(orappropriateelectrophoresisbufferifyou\"reusinganotherbufferforelectrophoresis).Load1to2.5¨microlitersonthe0.1mmthick,20cmx20cmcellulosethinlayerplatefromEM.Theamounttoloaddependsonhowmanycountsyouhave,butyoushouldnotloadmorethan30%ofthesample.Morethanthatwillcausethesampletosmearintheelectrophoreticdimension.![](\"dxy_expriment/201812/Electroorigins.gif\")
ForphosphorylatedLck,runpH8.9mapat1KVfor27min.inthe1stdimension.ForphosphorylatedLck,dochromatographyinPhosphochromobuffer.![](\"dxy_expriment/201812/or_ball.gif\")
Recently,chromatographyhastakenaslongas12hours.Itseemstovaryfrombatchtobatchofplates.Allyoucandoiswatchtheplatesandpullthemallwhenthefirsthasitsbufferfrontapproximately1cmfromthetop.For2liters n-Butanol 100ml Pyridine 50ml Aceticacid 50ml H2O 1800ml pH8.9buffer.(1%Ammoniumcarbonate)
Ammoniumcarbonate 20g H2O 2L pH1.9buffer.
For2liters 88%Formicacid 50ml Aceticacid 156ml H2O 1794ml Regularchromatographybuffer.
n-Butanol 97volumes Pyridine 75volumes Aceticacid 15volumes H2O 60volumes Phosphopeptidechromatographybuffer.
n-Butanol 75volumes Pyridine 50volumes Aceticacid 15volumes H2O 60volumes
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