LabelingTubulinandQuantifyingLabelingStoichiometryThisisageneralprocedureforcouplingmoietieswithreactivesuccinimidylesterstotubulin.Wehaveuseditsuccessfullytoderivatizetubulinwithsuccinimidylestersofbiotin,digoxigenin,andawiderangeofFluorochromessuchastetramethylrhodamine,X-rhodamine,fluorescein,OregonGreen,Cy3,Cy5andC2CF(bis-cagedcarboxyfluorescein).Theprocedureinvolveslabelingpolymerictubulin,therebyprotectingresiduesimportantformicrotubuleassembly.ThelabelingisperformedathighpHtooptimizethereactionwiththesuccinimidylestersandfunctionaltubulinisselectedafterthelabelingreactionbyoneormorecyclesofpolymerizationanddepolymerization. Backtoprotocols phosphocellulose-purifiedtubulin(~50mg=2-43mlaliquotsof5-10mg/mlPCfractions) DyestockinanhydrousDMSO(20-100mM) BRB80(1X):80mMPIPES,1mMMgCl2,1mMEGTA,pH6.8withKOH(generallymadeasa5Xstockandstoredat4¡C) HighpHCushion:0.1MNaHEPES,pH8.6,1mMMgCl2,1mMEGTA,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 Dye Excitation(nm) Emission(nm) emax=Extinctioncoefficientofdyeatitspeakwavelength e280/emax=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)I.Solutions&Supplies
emax(M-1cm-1) e280/emax Fluorescein 495 519 74,000 0.19 OregonGreen488 495 521 76,000 0.19 Cy3 550 570 150,000 0.08 Tetramethylrhodamine 550 576 95,000 0.21 X-Rhodamine 574 602 78,000 0.20 TexasRed 583 603 116,000 0.15 Cy5 649 670 250,000 0.05