LabelingTubulinandQuantifyingLabelingStoichiometry2资讯分析...
来自 : 蚂蚁淘
ArshadDesai
Notes:
ThekeyvariableinMTspindownexperimentsisATP.UnderhighATPconditions,conventionalMAPsareselectivelyco-sedimentedwithmicrotubules.IntheabsenceofATP(orinpresenceofAMPPNPwhichinducesrigorbindingofmotorstoMTs),bothmotorsandMAPswillbindtoMTsandpelletwiththemicrotubules.ForMAPandmotoranalysis,Ioftensupplementtheextractwithexogenoustaxol-stABIlizedMTstoensurethatbindingsitesarenotlimitingandIamnotseeingcompetitioneffects.Foraunknownprotein,itisbesttotryavarietyofconditions.BelowisaprotocolforMAPpelletingunderhighATPconditions.IhavelistedthemodificationsforMAP/motorprotocolafterwards.
MAPpelleting:
- PrespinextractinTLA100.3at70Kfor20"at4deg.C.Transfersupetotubeonice.
- ExtractsmusthaveasourceofGTP.ForXenopuseggextracts,wesimplyaddanenergyregenerationmixtoextractsandtheextractmaintainsphysiologicalGTPlevels.Fordilutetissuecultureextracts,itisbesttosupplementtheextractwith0.5mMMgGTP.Thiscanbedonebeforeoraftertheprespin.
- Add2mMMgATPtoextracts,warmtoRTandaddtaxolto5uM.Mixwellandincubatefor2"-3"beforeaddinganadditional15uMtaxol(finalis20uMtaxol).Incubatefor20"-30"atRT-37deg.C.(WeuseXenopusextractsforwhichphysiologicaltemperatureisRT;fortissueculturecells,tubulinwillpolymerizebetterathighertemperatures.30-33deg.Cisagoodcompromiserangetobalancepolymerizationandproteolysis.Extractsmustbesupplementedwithapeptideproteaseinhibitorcocktailjustbeforewarminguptopreventexcessiveproteolysis).
- Layerthepolymerizedmixtureontoa1MsucrosecushioninBRB80containing0.5mMATP,10uMtaxolandproteaseinhibitorsandpelletinaTLS55at40Kfor20"at22deg.C.YoucanalsouseafixedanglerotorsuchasaTLA100.3orTLA100.4.IliketopelletMTsat100-150,000gfor20-30"inTLA100rotorsover30-40%glycerol/sucrosecushions.Forlargerultrarotors,increasethespeedand/ortimetoreachanequivalentclearingfactor.
- Savesupeforgel/blot,aspiratecushionwhilewashing2-3xwithBRB80andremoveasmuchofthecushionaspossIBLe(MTsformaclear,gelatinouspellet).
- Boilpelletinsamplebufferandanalyze.
MAPandmotorpelleting:
- donotaddextraATP.
- add2-5mMMgAMPPNPand/orsupplementtheextractwith15U/mlhexokinaseand20mMglucose(shouldtryallthreeconditions-onlyAMPPNP,onlyglucose-hexokinase,both).
- supplementextractwithtaxol-stabilizedMTsto0.2-0.3mg/mlfinalconcentrationafteraddingtaxoltowarmedupextractasabove.ThisconcentrationisforconcentratedXenopusextracts-coulduse0.1mg/mlfortissueculturecellextracts.
NoteonBuffers:
IhavedonethiswithextractspreparedusingBRB80andXB(10mMHEPES,pH7.7;100mMKCl,50mMsucroseand1mMMgCl2-aclassicallyMT-unfriendlybuffer)andfinditworkswithboth.IthinkitisbesttotryYourFavoriteBufferandBRB80side-by-sidetomaximizechancesofsuccess.IendedupusingXBsinceIwastryingtorelateMAPprofilestocellcycle-dependentMTdynamicsinXBextracts.Thebufferinthecushionisnotveryimportant.
NoteonTaxolstabilizedMTs:
- Dilutetubulinto2mg/mltubulininBRB80+1mMGTP+1mMDTTonice;incubate5".
- Warmto37deg.C,add1/100volof0.02mMtaxolinDMSO;5"at37deg.C
- Add1/100volof0.2mMtaxol;5"at37deg.C
- Add1/100volof2mMtaxol;10-15""at37deg.C.TheseMTscanbestoredatRTforupto1week.
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