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IsolationofMicrotubules(BovineBrain)

LEVELII

Materials

  • Freshlyremovedbovinebrain2
  • Wiresieve(teastrainer)
  • Microtubulebuffer(MTbuffer)
    • 0.1MMES(2-(N-Morphilino)ethanesulfonicacid)
    • 1mMEGTA(EthyleneGlycol-bis(-aminoethylEther)N,N,N",N"-tetraaceticacid)
    • 0.5mMMgCl_2
    • AdjustpHto6.4
  • 8MGlycerolinMTbuffer
  • Virtishomogenizer(orequivalent)
  • Refrigeratedcentrifuge
  • GTP
  • Bradfordproteinassay

Procedure3

  1. Removethemeningesandperipheralbloodvesselsfromthebrain.Pureethebrainbypushingitthroughawiresieveanddirectlyintoachilledbeaker.

  2. Homogenizethecerebralhemispheresofthebrainat4°Cinatissuehomogenizercapableofhomogenizingwithminimalsheerforce.4Usethemaximumspeedallowableforyourhomogenizerfor10seconds.Placethebrainhemispheresinthehomogenizerwithmicrotubulebufferataratioof0.5mlofMTbufferto1gofwetweightofbraintissue.

  3. Centrifugethecrudebrainhomogenateat19,600xgfor30minutesat4°Ctoremoveconnectivetissueandcellulardebris.

  4. Decantthesupernatantandrecentrifugethesupernatantat27,000xgfor45minutesat4°Cforfurtherclarification.

  5. Collect10mlofthesupernatantanddeterminetheproteincontentofyoursampleusingtheBradfordproteinassay(AppendixG).Usebovineserumalbuminorlysozymeforestablishingastandardcurve.

  6. Decanttheremainderofthecrudesupernatantintoachilledbeaker,andaddanequalvolumeof8MglycerolinMTbuffer.

  7. AdddryGTP(MW523)tothecrudesupernatanttomakea1.0mMfinalconcentrationofGTPandincubatethemixtureat37°Cfor30minutes.

  8. Centrifugethemixtureat100,000xgfor60minutesat25°C.Itiscrucialthatthetemperaturebemaintainedat25°C.Atalowertemperaturethemicrotubuleswillnotpolymerize(thusnopellet);andatahighertemperaturethetubulinmaybedegraded.

  9. RemovethepelletandresUSPendin40mlofcoldMTbuffer.Incubatefor30minutesat4°C.Occasionalhomogenizationinagroundglasshomogenizerwillfacilitatedepolymerizationofthemicrotubules.

    Forgoodtubulinpolymerization,aseriesofpolymerizationsanddepolymerizations,withsubsequentcentrifugecollectionsisrequired.Steps7through9shouldberepeatedaminimumof3times.

  10. WitheachsuccessivepolymerizationwithGTPandsubsequentcollectionoftheprecipitatedmicrotubules,repeattheproteindeterminationoneachsample.

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