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Recycling Tubulin 实验方法

来自 : 蚂蚁淘

RecyclingTubulinWe"recycle"tubulinfractionsstoredat-80¡CafterthePCcolumnandstoretherecycledtubulininsmallaliquotsforday-to-dayuse.WegenerallystorerecycledtubulininInjectionBuffer(IB)withoutfreeGTP.WedothisbecausedepolymerizationappearstobemuchbetterinIB,IBisidealformicroinjections/addingtubulintoextracts,andtheabsenceoffreeGTPmakespolymerizationwithGMPCPP,averyusefulGTPanalogthathas~5-10XloweraffinitythanGTPfortubulin,relativelystraightforward.

I.Solutions&Supplies
II.RecyclingProtocol

Backtoprotocols

I.Solutions&Supplies

5XBRB80:(1X=80mMK-Pipes,1mMMgCl₂,1mMEGTA,pH6.8withKOH;store5XBRB80stockat4¡C)10XIB:500mMK-glutamate;5mMMgCl₂,(pHof1X~7.0;store10Xstockat-20¡C;seelabelingsectionfordetailsonmakingupIB)Cushion:60%glycerolin1XBRB80(warm=37¡C)100mMGTPGlycerolSmallDounce(2ml)50.2Tirotor(warm=37¡C)

TLA100.2or100.3rotor(cold=2-4¡C)II.RecyclingProtocol1.Thaw3-43mlPCcolumnfractionsat37¡C.Transfertoiceandmixina50mlconical.2.AddBRB80to0.5X,4mMMgCl₂and1mMGTP.ThisisassumingthatthetubulinaliquotsareinCB(50mMK-Pipes,pH6.8;1mMEGTA;0.2mMMgCl₂).Thus,thefinalbuffercompositionis90mMK-Pipes,pH6.7,1.5mMEGTA,4.2mMMgCl₂,1mMGTP.Mixbyswirlingandstoreonicefor5".3.Transferto37¡C.After2"addhalfvolumeofglycerol(Ifinditconvenienttopourintheglycerolonabalance,usingdensityforglycerol=1.26g/ml).Mixtheglycerolinbygentlevortexing.Incubateat37¡Cfor40".4.Layeronawarmcushion(10-14ml)intwo50.2Ti(orequivalent)tubes.Spinfor45"at40Kat35¡Cina50.2rotor.5.Aspiratesample,rinsesamplecushioninterfacewithwarmIXIB2-3times.Aspiratecompletelyandrinsethetubulinpellet2Xwithwarm1XIBtoremoveanyresidualglycerol.Dothiswhilethepelletisheldina37¡Cbath--filltube~half-waywithwarmIB(rinsingsidesandpelletwell),thenaspirateandrepeat.ThisgreatlyreducesglycerolandGTPcontaminationinthefinaltubulinstock.6.ResUSPendpelletin1-2mloficecoldIB(theexactvolumewilldependontheconcentrationandpolymerizABIlityofthetubulininthecolumnfractions).TransferthechunkypelletinIBtoanice-cold2mldounce.Gentlydounceonicetobreakupthechunks.Incubateonicefor30".Duringthiscolddepolymerization,keepdouncinggentlyevery2"-3".Gentlesonicationcanalsobeusedtobreakupthepellets.7.Spin90KinTLA100.2orTLA100.3rotorat2¡Cfor10"-15"toclarifythedepolymerizationmix.8.CollectsupernatantoniceandmeasureA₂₈₀ofa1/100dilutioninIB.Calculateconcentrationoftubulinusinganextinctioncoefficientat280nmof115,000M^-1cm^-1.Freezein10-50µlaliquotsinliquidnitrogenandstoreat-80¡C.(Note:1mg/mltubulin=10µM)