I.Solutions&Supplies

phosphocellulose-purifiedtubulin(~50mg=2-43mlaliquotsof5-10mg/mlPCfractions)

DyestockinanhydrousDMSO(20-100mM)

BRB80(1X):80mMPIPES,1mMMgCl₂,1mMEGTA,pH6.8withKOH(generallymadeasa5Xstockandstoredat4¡C)

HighpHCushion:0.1MNaHEPES,pH8.6,1mMMgCl_2,1mMEGTA,60%(v/v)glycerol

LabelingBuffer:0.1MNaHEPES,pH8.6,1mMMgCl₂,1mMEGTA,40%(v/v)glycerol

Quench:2XBRB80,100mMK-Glutamate,40%(v/v)glycerol

LowpHcushion:60%(v/v)glycerolin1XBRB80

10XIB(InjectionBuffer):500mMK-Glutamate,5mMMgCl₂(pHof1X~7.0)

50.2Tirotor(warm=37¡C)

TLA100.4orTLA100.3andTLA100.2rotors

SmallDounce(2ml)

Note:1MHEPES,pHto8.6withNaOHandstoreat-20¡C

2MK-Glutamate-dissolveglutamicacidto2M,carefullypHwithKOHsuchthat50mMhasapH~7.0andstoreat-20¡C

(Allbuffersforlabelingcanbestoredindefinitelyat-20¡C;dyestocksarebestpreparedfreshfrompowderthathasbeenstoredanhydrouslyat-20¡C;residualdyesolutioncanbestoredat-20¡Cor-80¡Cunderanhydrousconditions)

II.LabelingProtocolTheproceduredescribedbelowcanbescaleddownifdesired.Itisessentialtoperformallstepsinvolvingcageddyesunderasafelightinaroomwell-shieldedfromlight.Apieceofredacetatesheettapedoveradimlylitlampisadequateasasafelight.Otherdyelabelingscanbedoneunderroomlight,minimizingexposureduringincubationsbyusingfoil.

1.Thaw2-4PCcolumnfractions(30-60mgtubulin)andaddBRB80to0.5X,MgCl₂to3.5mM,GTPto1mMandstoreonicefor5".Transferto37¡CandaddDMSOto10%finalintwosteps,mixinggentlybutthoroughlyandincubateat37¡Cfor30min.Alternatively,addhalfvolumeofglyceroltopromotepolymerization.Inaside-by-sidecomparison,forreasonsthatarenotclear,usingDMSOinsteadofglycerolforthefirstpolymerizationstepappearstoincreasethelabelingstoichiometryby~25%forC2CF-SNHSester.Forlabelingwithrhodamine(tetramethylrhodamineNHSester)andX-rhodamine,wegenerallyuseglycerolpolymerization.

2.Layerpolymerizedtubulinonto20mlwarmHighpHCushionintwo50.2Titubes.PelletmicrotubulesinaBeckmanultracentrifugeina50.2Tirotorat40Kfor45"at35¡C.

3.Aspiratethesupernatantabovethecushionandrinsethesupernatant-cushioninterfacetwicewithwarm(37¡C)LabelingBuffer.AspiratethecushionandresUSPendthepelletusingacutofflargepipettipin1-2mlofwarmlabelingbuffer.TakecaretokeepthetubulinwarmduringtheresuspensionandcontinueresuspendingtillnochunksoftubulinarevisIBLe.Thisisthemostpainfulpartofthelabelingprocedure.

4.Add10-to20-foldmolarexcessofthedyetotubulin.Estimatethetubulinconcentrationassuming~70%recoveryofthestartingtubulin.FordyessuchasCy5andCy3,usea5-packforlabeling~25mg.Formostdyeswelabelfor30"-40"at37¡C.ForC2CF-SNHS(cagedfluorescein),wehavefounditbesttoaddthedyeintwosteps(20"apart)andlabelfor60"at37¡C.Afteraddingthedyestock,gentlyvortexthemixtureevery2"-3"duringthecourseofthelabeling.

5.AtendoflabelingincubationaddanequalvolumeofQuenchtothelabelingreactionandmixwell.Incubatefor5".

6.LayerthequenchedlabelingreactionontotwoTLA100.3(orTLA100.4)tubescontaining1.5mlofLowpHCushion.Spinat80Kfor20minat35¡CinaTLA100.3orTLA100.4rotorinaBeckmanTLA100ultracentrifuge.

7.Aspiratethesupernatantabovethecushionandrinsethesupernatant-cushioninterfacetwicewithwarm1XBRB80.Aspiratethecushionandresuspendthepelletusingacutoffpipettipin1mlofice-cold1XIB.Transferresuspendedchunksofthepellettoasmallice-colddounce(1or2mlvolume)inanice-waterbath.Resuspendthepelletbygentledouncingtillthesuspensionisuniform.Continuedouncingintermittentlyforatotaltimeof30minat0¡C.

ColdIBseemstopromotemorerapiddepolymerizationthanBRB80;therefore,weuseIBinthedepolymerizationstepforalllabelingprocedures.Forsmallscalelabelingsthepelletcanberesuspendeddirectlyinthecentrifugetubeandsonicatedgentlyusingamicrotipsonicatortospeeddepolymerization.

8.SpinthedepolymerizedtubulininaTLA100.2(orTLA100.3)rotorat80Kfor10"at2¡C.

9.Recoverthesupernatantfromthecoldspin,addBRB80to1X(froma5Xstock),MgCl₂to4mM,GTPto1mMandincubateonicefor3".Warmto37¡Cfor2",add1/2volumeofglycerol(33%v/vfinal),mixwellandpolymerizeat37¡Cfor30min.

10.Layerthepolymerizationreactionona1mlLowpHCushioninaTLA100.3tubeandpelletthemicrotubulesat80KinaTLA100.3rotorfor20"at37¡C.

11.Aspiratethesupernatantabovethecushionandrinsethesupernatant-cushioninterfacetwicewithwarmIB.Aspiratethecushionandrinsethepellettwicewith1mlwarmIBtoremoveanyresidualglycerol.Resuspendthepelletusingacutoffpipettipin0.2-0.3mloficecoldIB.Thispelletshouldresuspendeasily.Incubateat0¡Cfor20to30min.

12.SpinthedepolymerizedtubulininaTLA100orTLA100.2rotorat80Kfor10"at2¡C.Recoverthesupernatant,quicklyestimatethetubulinconcentration,adjustwithIBifdesiredandfreezein3-5ulaliquotsinliquidnitrogen.Wegenerallyaimforafinaltubulinconcentrationof5-15mg/ml(50-150µM).Carefuldeterminationoftubulinconcentrationandlabelingstoichiometrycanbeperformedasdescribedbelow,afterthetubulinhasbeenaliquotedandfrozen.C2CF-tubulinshouldbestoredat-80¡Cinafoil-wrappedbox.

III.QuantifyingTubulinConcentrationandLabelingStoichiometryTodeterminethetubulinconcentrationandstoichiometryoflabeling,dilutethelabeledtubulin1/50-1/100inIBandobtainawavelengthspectrum.Calculatethemolarconcentrationofdyebyusingtheabsorbanceatthepeakwavelengthandtheextinctioncoefficientprovidedbythedyemanufacturer.DeterminethetubulinconcentrationbyfirstsubtractingoutthecontributionofthedyetotheA₂₈₀andthenusinganextinctioncoefficientof115,000M^-1cm^-1.SectionVprovidesalistofextinctioncoefficientsandA₂₈₀absorbance(relativetoabsorbanceatpeakwavelength)forcommonlyuseddyes.NotethattheabsorbanceoffluoresceinispH-dependentandconjugateswithfluoresceinshouldeitherbedilutedintoahighpHbuffer(~8.8-9.0)orthevaluemeasuredatpH7.0multipliedby1.2.

Anexampleofcalculatingconcentrationandstoichiometryfortubulinlabeledwithtetramethylrhodamine(TMR)NHSester:

Tubulinconcentration=[(A₂₈₀-ContributionofdyetoA₂₈₀)xDilutionFactor]/Extinction

coefficientoftubulinat280nm

TMRconcentration=(A₅₅₅xDilutionFactor)/ExtinctionCoefficientofTMRat555nm

LabelingStoichiometry=TMRconcentration/Tubulinconcentration

Awavelengthspectrumof1/100dilutionofthefinallabeledtubulinproductgavethefollowingabsorbancevalues:

A₂₈₀=0.23;A₅₅₅=0.20

Therefore,Tubulinconcentration=[{0.23-(0.2x0.2)}x100]/115000=165µM

TMRconcentration=[0.20x100]/95000=210µM

LabelingStoichiometry=210/165=1.3

TodeterminetheconcentrationandlabelingstoichiometryofC2CF-tubulin,theC2CFmustbefirstuncagedtofluorescein.Todothis,dilutethelabeledtubulin1/50to1/100inIB+2mMDTTinanEppendorftube.PuttheeppendorftubeonahandheldUVlamp,coverwithfoil(shinysidedown)andexposetolongwavelengthUVfor30".Obtainawavelengthspectrumfrom200to600nmafterthe30"activation,usingIB+2mMDTTexposedtoUVinparallelasablank.Assuminga100%efficiencyfortheuncagingreaction,theconcentrationofC2CFcanbecalculatedfromthespectrumafteractivationasfollows:

ConcentrationofC2CF=(A₄₉₅xDilutionFactorx1.2)/74000

(Thefactorof1.2correctsforthepHdependenceoftheabsorptionspectrumoffluorescein)

IV.UsingLabeledTubulinsa)Microinjectionintocells/additiontoextracts:Formicroinjections,wedilutethetubulininIBto2-5mg/ml,clarifybycentrifugationandinject~1/10ofcellvolume.Forfrogextractstudies,weaddlabeledtubulinto1/40-1/200thoftheextracttubulinpool(~20µM).

b)Preparationoffluorescentmicrotubulesubstratesorformonitoringpolymerizationanddynamicsofpuretubulin:Weuseamixtureoflabeledandunlabeledtubulinforpolymerization.Theratiooflabeledtounlabeledwilldependontheparticularapplicationandonthebrightnessofthelabeledtubulin.Labeledtubulins,especiallythoselabeledtohighstoichiometry,exhibitverydifferentpropertiesfromunlabeledtubulin.Therefore,weusethehighestratioofunlabeledtolabeledtubulinthatprovidessignalintensitysufficientforaparticularexperiment.V.PropertiesofFluorescentDyesUsedforTubulinLabeling

e_max=Extinctioncoefficientofdyeatitspeakwavelength

e₂₈₀/e_max=Absorbanceofdyeat280nmasafractionofitsabsorbanceatitspeakwavelength

5(and-6)carboxyfluoresceinsuccinimidylester(MolecularprobesC-1311)

OregonGreen488carboxylicacid,succinimidylester5-isomer(MolecularProbesO-6147)

5(and-6)carboxytetramethylrhodaminesuccinimidylester(MolecularProbesC-1171)

5(and-6)carboxy-X-rhodaminesuccinimidylester(MolecularProbesC-1309)

TexasRed-Xsuccinimidylester,mixedisomers(MolecularProbesT-6134)

Cy3-OSumonofunctionalreactivefluorophore(AmershamPA13100)

Cy5-OSumonofunctionalreactivefluorophore(AmershamPA13600)