Althoughmanyprotocolsfortubulinpreparationareavailable,theproceduredescribedbelowisthesimplestandhighestyieldingpreparationIhavedone.Theprotocolcallsfor3pigbrains,andshouldyield~60mgofpurifiedtubulin.Solutionsandreagents:100gP-11cellulosephosphatefibrouscationexchanger(WhatmanInc,CliftonNJ)6L0.1MHCl6L0.1MNaOH2L0.1MMgSO42L10xColumnBuffer~10L1xcolumnbuffer10MNaOH3fresh(<3hrsafterslaughter)pigbrains300mlHomogenizationbuffer(freshlyprepared)1LPMbuffer100mMMgATP1LPMGBuffer100mMMgGTP1Mdithiothreitol(DTT)Glutamicacid,sodiumsaltEquipment:WaringBlenderTemperaturecontrolledultracentrifuge(BeckmanL7-55)withtheequivalentofaBeckman50.2TirotorAtleast2431.5mlthick-walledpolycarbonateultracentrifugetubes(Beckman#336091)withscrewcaps(Beckman#338906)37oCwaterbath30mlDounce"A"glasshomogenizer2Lscinteredglassfilterfunneland2LsidearmErlenmeyerflaskAmicon(Danvers,MA)44mmx250mm#95240orequivalentadjustablevolumecolumnforlowpressureliquidchromatographyPeristalticpumpFractioncollectorUVmonitororBradfordreagent(Biorad)DAY1:Preparationofthephosphocellulosecolumn.
- Weighout90gofWhatmanP-11phosphocelluloseandadditto2Lof0.1MNaOHina4LbeakerwhilemixingVERYgentlywithaglassrod.MixthesUSPensiongentlywiththerodfor5minutes,thenallowthesolidtosettlefor20min.Aspirateoffexcesssolution,andtransfertheremainingphosphocelluloseslurrytoa2Lscinteredglassfunnelona2LsidearmErlenmeyerflaskthatisconnectedtoavacuumline.Carefullyvacuumfiltertheremaining0.1MNaOHfromthephosphocellulose,BUTNEVERALLOWTHERESINTORUNDRY!
- Gentlyscrapethephosphocelluloseoutofthefunnel,andreturnittothe4Lbeaker,add2Lof0.1MNaOH,mixgentlyfor5min,andcheckthepHwithpHindicatorpaper.Repeatthemixingsettling,aspiration,andfilteringoftheresinasinstep1.IfthepHoftheslurrywasnotabove12,transferthephosphocellulosetothe4Lbeaker,add2Lof0.1MNaOH,andrepeatthemixing,settling,aspiration,andfilteringtreatmentasinstep1,leavingthewetphosphocelluloseinthefunnel.
- Rinse4Lofdistilledwaterthroughthephosphocellulosebyvacuumfiltration,again,neverlettingtheresinrundry.
- Transferthephosphocellulosefromthefunneltothe4Lbeakerandadd2L0.1MHCl.Mixgently,allowtheresintosettle,aspirateoffexcesssolution,andvacuumfiltertheresinasinstep1.Repeatthe0.1MHCltreatmentcycleuntilthepHofthephosphocelluloseslurryisbelow3.
- Rinsethephosphocellulosewith4Lofdistilledwaterbyvacuumfiltration.
- Transferthephosphocelluloseresintothe4Lbeakerandadd2Lof0.1MMgSO4.Mix,settle,aspirate,andvacuumfilterasinstep1.
- Transferthephosphocelluloseresintothe4Lbeaker,add2Lof10xcolumnbuffer.Mixgentlyfor10-15min,allowtheresintosettle,aspirateexcesscolumnbufferoff,andvacuumfilter.
- Transfertheresintothe4Lbeaker,add2Lof1xcolumnbuffer,mixgentlyfor5min,checkthepHoftheslurrywithapHmeter,andadjustitto6.6with10MNaOH.Allowtheresintosettle,aspirateexcessbuffer,andvacuumfilterit.Repeatthe1xcolumnbuffertreatmentcycle,adjustingthepHoftheslurryeachtimeto6.6with10MNaOH,untilthepHoftheslurryis6.6withoutadjustmentafterresuspensionandmixing.Allowtheresintosettlefor20min,aspirateoffbufferuntilthesettledresin:bufferratiois3:1(vol:vol).
- Pourthecolumn.Mixtheresingentlyuntilitisevenlysuspendedinthebuffer,thenrapidlypourtheslurryintoanempty44mmx250mmliquidchromatographycolumn(clampedtoasupportinthecoldroom)tofillittothetop.CoverthecolumnwithParafilm,andallowtheresintosettleforseveralhours.
- Fillthecolumnadjusterplungerwithcolumnbuffer,andVERYslowlyinsertitintothecolumn,beingverycarefulnottodisturbthephosphocelluloseresin.Astheadjusterplungerisinsertedintothecolumn,thecolumninlettubingthatisattachedtotheplungershouldbeimmersedina4Lreservoirofcolumnbuffer,andshouldexpelallairbubblesastheadjusterplungerisinserted.Tightenandsealtheadjusterplungerfittings,leavingtheinlettubeinthebufferreservoir.
- Attachtheoutlettubingtotheperistalticpump,andsetthepumptorunat0.25ml/mintoallowthecolumntopackproperlyforthenext~48hrswhileMTproteinisprepared.Awell-packedphosphocellulosecolumnshouldbeperfectlyevenincolorwithnoevidenceof"cracks"intheresin.Thebetterthephosphocellulosecolumnispacked,themoreconcentratedthepeakofelutionoftubulinprotein.
DAY2:CyclingpreparationofMTprotein.
- Keepthebrainsinanevacuatedplasticziplockbagburiedinicefromthetimeofslaughterduringtransportbacktothelaboratory.
- Ina4oCcoldroom,carefullyandthoroughlyremovethemeningesandanyblood-redtissuefromthesurface,stem,andwithinthefoldsofeachbrain.Dothisbybothpickingtissueawaybyhandandwipingwithkimwipes.Thedrywipewillsticktothemeninges,peelingawaythedeepredmembranefromthepinkishgreynervoustissueunderneathasthewipeisgentlydrawnacrossthesurfaceofthebrain.
- Cutthecleanedbrainsinto2-3cm2cubesandweighthetissue.TransferthetissuetoaWaringblenderandadd0.5ml/gfreshlypreparedHomogenizationBuffercontaining1mMMgATP.
- Homogenizethetissuebyblending5sonhighspeedandthen45sonlowspeed.Thisshouldresultinasuspensionwiththecolorandconsistencyofastrawberrymilkshake.
- Usinga50mlSEROlogicalPipettewith1/3ofthetipcutoffandapipettebulbwithstrongsuction,transferthebrainhomogenatetoseveral31.5mlpolycarbonateultracentrifugetubes,notethehomogenatevolume,andpairthetubesbyweight(to0.01g).Discardanyextrahomogenatethatdoesnotfitintoafullrotorofcentrifugetubes.Tubulinissensitivetoproteasesandeasilydenatured,andkeepingextrahomogenateforonehourduringthefollowingcentrifugationstepdoesnotsignificantlyincreasethefinalyield.
- Centrifugethehomogenateat100,000xg(29,000rpm)for60minat4oCina50.2Tirotortoremoveundisruptedtissuefromcellcytosol.
- Atroomtemperature,carefullycollectthecytosolicsupernatantsfromthetubeswithapipette,pooltheminagraduatedcylinder,dilutethetotal1:1withPMG,andaddMgGTPto0.2mMtopromoteMTpolymerization.Disburseinto31.5mlultracentrifugetubesandpairthembyweight.
- Immersetheportionofthetubescontainingthecytosolina37oCwaterbathandallowMTproteintopolymerizeintoMTsbyincubatingfor45minutes.Duringthispolymerizationincubation,warmtheultracentrifugeand50.2Tirotorto25oC.
- PelletMTsfromthecytosolbycentrifugationat100,000xg(29,000rpm)for45minat25oCina50.2Tirotor.
- Inthecoldroom,discardthesupernatantandresuspendtheMTpelletin1/5thevolumeofhomogenateinPMbuffercontaining0.2mMGTP.Todothis,putafewmlsofresuspensionbufferintoeachcentrifugetube,aswellasintoasmallglassDouncehomogenizer(onice).Scrapeoutthestickypelletswitharound-endedweighingspatulaandtransferthemintothebufferinthehomogenizer.Resuspendanypelletremainingintheultracentrifugetubesbytrituration,andtransferittothehomogenizer.Homogenizethepelletsby5-10passeswithatype"A"pestle.DisbursetheresuspendedMTsinto31.5mlultracentrifugetubesandpairthembyweight.
- IncubatetheresuspendedMTsonicefor30minuteswithgentlemixingevery5mintoallowforMTdepolymerization.Duringthistimechilltheultracentrifugeand50.2Tirotorto4oC.
- ClarifytheMTproteinbycentrifugationat100,000xg(29,000)rpmfor45minat4oCina50.2Tirotor.
- Atroomtemperature,collectthesupernatantcontainingtheMTprotein,diluteit1:1withPMG,addGTPto0.2mM,disburseitinto31.5mlcentrifugetubes,andpairthembyweight.
- ImmersetheportionofthetubescontainingtheMTproteinsolutionina37oCwaterbathandallowMTstopolymerizebyincubationfor45min.Duringtheincubation,warmtheultracenrtifugeand50.2Tirotorto25oC.
- PellettheMTsfrompolymerization-incompetenttubulinbycentrifugationat100,000xg(29,000rpm)for45minat25oCina50.2Tirotor.
- Inthecoldroom,discardthesupernatant.Add1mlofcolumnbuffercontaining0.5mMGTPtoeachtube,andbeingcarefulthatthebufferiscoveringthepellet,immersetheultracentrifugetubesintoliquidnitrogentofreezethepellets.Storethetubesat-80oCuntilthenextdayorwheneverphosphocellulosecolumnpurificationoftubulinistobecarriedout.
DAY3:PhosphocellulosecolumnpurificationoftubulinfromMTprotein
- Chillultracentrifugeand50.2Tirotorto4oC.
- Prepareandequilibratethephosphocellulosecolumnwithcolumnbuffercontaining0.5mMGTPand1mMDTT.Turnofftheperistalticpump,andiftheresinbedhassettled,carefullyloosenthesealsontheadjusterplunger,slowlyinserttheadjusterplungerfurtheruntilitbarelytouchesthetopoftheresin,andretightentheseals.Switchtheinlettubefromthereservoirof1xcolumnbuffertoa1Lreservoirof1xcolumnbuffercontaining0.5mMMgGTPand1mMDTT,beingcarefulnottointroduceanybubblesintotheinlettube.Turntheperistalticpumpbackonandadjustthespeedto1.8ml/min.Allowatleast300mlofbuffertobedrawnthroughthecolumnbeforeloADIngtheMTprotein.Duringthephosphocellulosecolumnequilibration,performsteps3and4below.
- ThawtheMTpelletsbyimmersingtheultracentrifugetubesina37oCwaterbathuntilthepelletsturnfromchalkywhitetocompletelytranslucentwhite.Assoonasthepelletsarethawed,placethetubesoniceimmediatelyandtakethemintothecoldroom.
- ResuspendthepelletsonicewithaDounceglasshomogenizerandtype"A"pestle(asinstep10ofpreparationofMTproteinabove)in~3xtheirvolumeof1xcolumnbuffercontaining0.2mMGTP.TransfertheresuspendedMTstoa31.5mlultracentrifugetube.AllowtheMTstodepolymerizebyincubationwithgentlemixingevery5minonicefor30min.
- ClarifytheMTproteinbycentrifugationat100,000xg(29,000rpm)for45minat4oCina50.2Tirotor.
- Inthecoldroom,collecttheclarifiedMTproteinsupernatantandaddMgGTPtoafinalconcentrationof0.5mM(anadditional0.3mM)andDTTto1mM.
- Aftercolumnequilibration,switchthecolumninlettubefromthebufferreservoirtotheclarifiedMTprotein.LoadtheMTproteinontothecolumnat1.8ml/min.WhenalloftheMTproteinisloaded,switchtheinletbacktothebufferreservoirandbegintocollect10mlfractions.Tubulinwillpassthroughthecolumnandcomeoffafter~100mls,whileMTbindingproteinswillremainboundtothephosphocelluloseresin.
- MonitortheelutionoftubulineitherwiththeUVmonitorbyabsorptionat280nmorbyadding100ulofeachaliquotto1mloffreshlypreparedBradfordreagent(Biorad)andlookingforbluecolor.Aseachaliquotcomesoff,addMgGTPtoafinalconcentrationof1mM(anadditional0.5mM).Poolfractionscontainingtubulin(usually~100ml).
- Atroomtemperature,add0.186g/mlglutamicacid(sodiumsalt)tothetubulinsolutionandstirslowlyuntilitisdissolved.Disbursethesolutioninto31.5mlultracentrifugetubesandpairthembyweight.
- Immersethecentrifugetubestotheleveloftheliquidwithinina37oCwaterbathandincubatefor30mintoallowMTstopolymerize.Duringthistime,warmthe50.2Tirotorandultracentrifugeto25oC.
- IsolatetheMTsfrompolymerization-incompetenttubulinbycentrifugationat100,000xgfor30minat25oCina50.2Tirotor.
- Inthecoldroom,resuspendtheMTpelletsonicewithaDounceglasshomogenizerasabove,in3xtheirvolumeofPMbuffercontaining0.5mMMgGTP.IncubatetheresuspendedMTsonicefor30mintoallowMTdepolymerization.Duringthistime,determinethetubulinconcentrationbymeasuringtheabsorbanceat280nm(besuretoblankthespectrophotometeragainstthesamePMbuffercontaining0.5mMMgGTP)usingtheextinctioncoefficientoftubulin=115,000Mol/cm([tubulin]=(A280xdilutionfactor)/extinctioncoefficient)).AddPMbuffercontaining0.5mMMgGTPtoadjustthefinalproteinconcentrationto45uM.
- Disburseintoseveral1mlaliquots(stockaliquots)andseveral50ulaliquots(tobeuseddirectlyintheMT/Organellemotilityassays),freezebyimmersioninliquidnitrogen,andstoreat-80oCuntiluse.*=Aspublishedin...Waterman-Storer,C.M.(Inpress)Microtubule/organellemotilityassays.In:CurrentProtocolsinCellBIOLOGy,J.S.Bonifacino,M.Dasso,J.B.Harford,J.Lippincott-Schwartz,andK.M.Yamada,eds.JohnWiley,NY.
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