I.TubulinPrepOutline

Pre-Prep:

1.Callslaughterhouseandrequestfreshbrainstobepickedupthemorningofprep

2.Pourandequilibratephosphocellulosecolumn

3.Makebuffers

4.Ensurethatreagents,suchasATPandGTP,arepresentinsufficient

amountfortheprep

5.Signupforcentrifugesandrotorsandgathercentrifugetubes,

blendersandmotorizedhomogenizer/dounce

6.Preparecoolerfortransportingbrainsnightbeforeprepandorganize

coldroomformorningmayhem

PrepDay:

7.Removemeninges,brainstemsandbloodclots,weighand

homogenizebrainsinblenders

8.Clarifyhomogenateandusesupernatantfor1stpolymerizationcycle

9.Collect1stcyclepolymerfractionbycentrifugation

10.Depolymerize1stcyclepolymerbyhomogenizationat0-4⁰C

11.Clarifydepolymerizationmixandusesupernatantfor2nd

polymerizationcycle

12.Collect2ndcyclepolymerfractionbycentrifugation

13.Depolymerize2ndcyclepolymerbyhomogenizationat0-4⁰C

14.ClarifydepolymerizationmixandloadsupernatantontoPC

column

15.CollectflowthroughfromPCcolumn,aliquotandfreezeat-80⁰C

II.Buffers&Nucleotides

PB(Pipes/PolymerizationBuffer):0.1MK-Pipes,pH6.8,0.5mMMgCl₂,2mMEGTA,0.1mMEDTA,0.1%b-mercaptoethanol,1mMATP.Need8litersincoldroom(AddATPandBMEjustpriortobeginningtheprep)CB(ColumnBuffer):50mMK-Pipes,pH6.8;1mMEGTA;0.2mMMgCl₂.Need~25litersforequilibration,runningandstorageofPCcolumnCB+1MKCl:Need~10litersforprewashingandelutingthePCcolumnTomake1Lof10XCB:151.2gramsPIPES,freeacid3.8gramsEGTA2mlof1MMgCl₂pHwithKOHtopH6.75,andbringupto1liter.CheckpHat1Xis6.7Make3.5litersof10XCBfor10-12brainprep.GTP:SigmaTypeIIS-#G-8752ATP:SigmaGrade1-#A-2383Glycerol:2-3Lprewarmedto37¡C(storeovernightin37¡Cincubator)

III.Pouringa1LPhosphocellulose(PC)Column

Resin:WhatmanP11CellulosePhosphate--fibrouscationexchanger(1gramofPCswellstoabout4mlpackedresin)Summary:Topoura1Lcolumn,startwith220gramsdryresindividedinto5aliquotsof44grams.Treateachaliquotwithacid/baseina2Lbeakerasdescribedbelow.OlderproceduresdescribedtheuseoflargeBuchnerfunnelstorapidlyremovetheacid/base.However,gentlestirringoftheresinwithaplastic/glassrodtosUSPenditina2Lbeaker,followedbysettlingoftheresinfor5"bygravityhasworkedwellforus.Thismethodalsoincorporatesde-finingoftheresinintotheacid/basecyclingprotocol.Afteracid/basetreatmenttheresiniswashedwell,packed,treatedwithBSAtoblockirreversIBLebindingsitesandequilibratedforuse.Solutions&Supplies:220gramsPhosphocellulose5L0.5NNaOH5L0.5NHCl13L0.5MK-Phosphate,pH6.85LddH₂052Lbeakers12LCB+1MKCl20LCB300mlof30mg/mlBSAinCB(filtered)1-1.5Lcleanedcolumnhousing2stirringrods2aspiratorswithlargetrapsPeristalticpump10mlplasticpipetsasinletsforperistalticpump

ColumnPreparationProcedure:1.Pour1L0.5NNaOHinto5x2Lbeakers.Add44gramsPCtoeachbeakerstirringgentlywitharoduntilthePCiswettedandanevenslurryispresent.Letstandatroomtemperaturefor5".2.Aspirateoffsupernatant,includingfines,andquicklyadd1L0.5MK-phosphatetoneutralize,gentlymixingwitharod.CheckthatpHis~7andletstand5".3.Aspirateoffsupernatant,add1LddH₂0andgentlystirtoresuspendsettledresin.4.Allowtheresintosettle.5.Aspirateoffsupernatant,add1L0.5NHCl,gentlystirtoresuspendandwait5".6.Aspirateoffsupernatant,add1L0.5MK-phosphate,stirandcheckpHis7.7.Afterresinhassettled,aspiratesupernatantandcombinealltheresinina4Lbeaker.Usetheremaining0.5MK-phosphatetowashtheresinbyresuspending,lettingsettleandaspiratingthesupernatant.8.Wash3x1LCB+1MKClasdonein7.9.Inthecoldroom,pourtheresuspendedresinintothecolumnhousing(withamarkapprox.50cmhighina5cmIDhousing)andpackbypumpingfromthebottom(i.e.theperistalticpumpis"sucking"bufferfromthebottomofthecolumnanddepositingitintoawastejug).Packat45ml/hour/cmcross-sectionalarea.Fora5cmdiametercolumnthisis~880ml/houror~14.5ml/min.Afterresinispacked,switchtopumpingfromthetop.Run7LofCB+1MKClthroughthecolumnat5-10ml/min.10.Washwith10LCB.CheckconductivitytoensurethatalltheKClisgone.Theresinmayexpandasthesaltiswashedoutsomakesurethereisalargebufferheadontheresinbed.11.Load300mlof30mg/mlBSA(FractionV;filtered)inCB,followwith700mlCBandstopthecolumn.Leavethecolumnsittingfor2hrduringwhichtheBSAblocksirreversiblebindingsitesontheresin--thisisveryimportantthefirsttimeacolumnisusedtopreventlossofthetubulin.12.Washthecolumnwith2LCB+1MKCltoeluteBSAthatisnotirreversiblybound.13.Washcolumnwith10LofCB.Thecolumnisnowreadyforuse.

IV.Brains

Itisessentialtogetfreshbrains(theyshouldbewarmwhenhandedtoyouattheslaughterhouse)--yieldsdeclinesignificantlyifthebrainshavebeenstoredforawhileafterremoval.Frozenbrainsdonotworkforpreparingtubulin.Thebestprepshavebeendonewithfreshlyremovedbrainstransportedinanice-filledcoolertothelabwithin1-2hoursofremoval.Fortransporting10-12brainsfromtheslaughterhouse,weuse2size16Colemancoolerscontaining3litersofcooled1.5%(w/v)NaCltowhichonelargebagofpartyiceisaddedonthewaytotheslaughterhouse.

V.Centrifuges&Rotors

6SorvallRC-5Corequivalentlowspeedcentrifuges

6GSAorequivalentrotors(cold)

4Beckmanultracentrifuges

4Type19rotors(warm)

2Type35rotors(warm)

2Type45Tirotors(cold;needtowarmuponeafter1stcolduse)

1Type50.2Tirotor(cold)

cold=4¡C(putovernightincoldroom)

warm=37¡C(puttherotorsovernightinalargebacterialshakersetto37¡C)

VI.Protocol

1.Inthecoldroom,removemeninges(membranesurroundingthebrain;bestdonebyusingpapertowelsto"blot"thebrainsurface),bloodclots,andbrainstems;weighthebrainsandhomogenizewithequalvolumeofPBfor3x15sinaWaringblender.2.Collecthomogenate(~8-9liters),transferinto36GSAbottlesandspin90"at12KinaGSArotorat4¡C.3.Collectsupernatantandtransfer1litertoa1.8LglassFernbachflaskthathas500mlof37¡Cglycerol.Add0.1mMGTP,0.5mMATP,and3.5mMMgCl₂(thisgives0.1mMGTP,1.5mMATPand4mMMgCl₂final).TheATPandGTPareaddedassolids.Holdtheflaskinawarmwater-filledsinkandswirlconstantlytodissolvethesolidsandtomixintheglycerol.Transfertoa37¡Cbath,monitortemperatureofmixtureusingacleanThermometerandpolymerizefor60"afterthetemperatureofthesamplehasreached32¡C.Theapproachto37¡Ccanbeacceleratedbyswirlingtheflaskinalargehotwater(~50¡Ctemperature)reservoir--constantswirlingisessentialinthiscasetodispersetheheatevenlyandcaremustbetakentoavoidoverheatingthemixture.4.TransferthepolymerizationmixturetoType19bottles,andspinfor2.5hrsat19Kin4Type19rotorsat35¡C.UseanadditionalType35at17.5Kfor2.5hrsifnecessary.Attheendofthespinsetcentrifugesto4¡C.5.Decantanddiscardsupernatant.Inthecoldroom,resuspendthegelatinouspelletsinPBaimingforafinalvolumeof~700-800ml.Weuse~40-50mlfor3tubes,sequentiallyremovingthepelletsfromeachtubeusingaplasticscraperandmakingsurethatalltubesgetrinsedonceortwiceafterthemajorityofthelargegelatinouspellethasbeenremoved.Tohomogenizethechunkypelletresuspension,weuseaYamato"pour-through"continuousflowhomogenizer--thisisadevicethatdrivesamotorizedteflonpestleinafunnelshapedglassbarrel.Mixturespouredontopgethomogenizedbythepestleastheytravelthroughthemiddleofthebarrelandcomeoutthebottom.WeYamatothechunkypelletstilltheresuspensionisasmoothyellowliquidof~700-800mltotalvolume.Afterallthepelletsarehomogenized,wedepolymerizeonicefor~30"duringwhichwecontinueYamatoingthemixtureonceevery3"-5".AlargemotorizedteflondounceorlargetipsonicatorcanbeusedasalternativestotheYamatoforresuspendingthechunkypellets.CheckproteinconcentrationbyBradfordusingBSAasastandard.If>20mg/ml(whichisunlikely),diluteto20mg/ml.6.Spinthedepolymerizationmixture30"at35Kin2Type45rotorsat4¡C.Attheendofthespinsetthecentrifugesto35¡C.7.Decantsupernatantintoa1Lgraduatedcylinderincoldroomandmeasurevolume.Pourintoa1.8LFernbachflask,addhalfvolumeof37¡Cglycerol,solidGTPto0.5mMfinalandMgCl₂to4mMfinal(additional3.5mM).Setuppolymerizationasdescribedin3.above.Polymerizefor40"aftertemperatureofmixturehasreached32¡C.8.Spinthepolymerizationmixtureat35Kat35¡Cfor1hrin2Type35s+1Type45.Makesureonechilledcentrifugeisavailableforthenextspin.9.Discardsupernatantandresuspendpelletsinafinalvolumeof~100-150mlofCB,asdescribedabovein5.ProteinconcentrationbyBradfordshouldnotbemorethan25mg/ml.Depolymerizeonicefor40"andthenspinthedepolymerizationmix30"at40Kina50.2Tirotorat4¡C.10.Collectsupernatant,measureconcentrationbyBradfordandloadontothePCcolumn(approx.50cmhighX5cmID=~1L;Flowrate=6ml/min).Afterthesampleisloadedand~150mlbufferhasflowedthroughstartcollectingfractions.Theelutedtubulinwillbeapparentbyitsslightyellowishtinge.MeasureconcentrationbyBradfordusingBSAasastandardandpoolsuchthatthefinalconcentrationisbetween5-10mg/ml.Mixpoolonice,make3mlaliquotsin5mlsnapcappolypropylenetubesandfreezeinliquidnitrogen.Storefrozenaliquotsat-80¡C.Theentireprocedure,fromtimeofarrivalofbrainstillfreezingofthetubulinwilltake~17-18hours.Thenextday,run3volumesofCB+1MKCltoeluteMAPsfromthePCcolumn(thesecanbecollectedifdesired),andthenequilibratecolumnintoCB+0.1%azideforstorage.Phosphocellulosewilllosecapacitywhenstoredwet--thiscanbereducedbystorageinaphosphatebuffer(50mMphosphate,pH7with1mMEGTAand0.2mMMgCl₂)containing0.1%azide.