LEVELII

Materials

• Freshlyremovedbovinebrain2
• Wiresieve(teastrainer)
• Microtubulebuffer(MTbuffer)
  • 0.1MMES(2-(N-Morphilino)ethanesulfonicacid)
  • 1mMEGTA(EthyleneGlycol-bis(-aminoethylEther)N,N,N",N"-tetraaceticacid)
  • 0.5mMMgCl
  • 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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