1.	Obtainpigbrainsandmeasuretheweight.Keeponice.

2.	Workingrapidly,removemeningesandbloodvesselswithKimwipesat4°C.Prepareenoughspaceincoldroomfor2-3personsworkingtogetheratthisstep.(Needbenchcover,trashbag,Waringblender,300mlbeakerinicebucket.)

3.

Mincethetissuesrapidlywithscissorsthathavebeenrinsedwithethanol,thenaddPM-4Mbufferataratioof100mlper100goftissue(saveathirdtorinsetheblenderjar),andhomogenizethetissueinaprecooledWaringblenderatthelowestspeedfor40sec.TransferthemixtureintoanewbeakerandrinseouttheblenderjarwithsmallvolumeofthePM-4Mbuffer.

4.	Centrifugethehomogenatefor15minat4°Cat9500rpmintheSorvallSS-34rotor(6500g).

5.	Decantthesupernatantsinto30mlBeckmanultracentrifugetubesoniceandcentrifugeat30000rpminaBeckmanTi70rotor(96000g)for75minat4°C.

6.	Decantthesupernatantsintoagraduatedcylinderandcheckthevolume(V1).

7.	Add1MGTPtoafinalconcentrationof0.5mMandmixgentlybyinvertingthecylinderserveraltimes.Transferintonew30mlultracentrifugetubes.

8.	Balancethetubesandincubatefor45mininawaterbathat34°C(thesolutionatthisstageispinkish).Duringtheincubation,warmtheTi70rotorwithtapwaterandsettheultracentrifugetemperatureto27°C.

9.	Centrifugethetubesfor60minat27°Cat30,000rpmtopellettheassembledmicrotubules.

10.	Atroomtemperature,decantthesupernatantsfromthefirmandslightlyopalescentpellets.

11.	ResUSPendthepelletsinavolumeof0.25xV1ofice-coldPMbuffer.

12.	Removethepelletsfromthebottomsofthetubebygentlyscrapingwithaglassrod.Pourtheresultingsuspensionintoanice-coldDouncehomogenizerandhomogenizeusing5gentlestrokesofthepestletoproduceauniformsuspension.

13.	Incubatethesuspensiononicefor30mintodepolymerizemicrotubules.

14.	Transferintoice-coldultracentrifugetubesandcentrifugeat30000rpmat4°Cfor60mininaBeckmanTi70rotor.

15.	Carefullyremovethesupernatantsfromthepellets,whicharesometimesloose,andpoolinagraduatedcylinder.Notethevolume(V2).

16.	AddanequalvolumeofPM-8M,mixgentlyandstoreat-20°C.Thetubulinatthisstagecanbestoredaslongas72hrs.

Nextday

17.	Asecondcycleofpolymerization/centrifugationanddepolymerization/centrifugationiscarriedoutbyfirstincubatingthemixtureat34°Casdescribedabove(steps8-10).Resuspendtheresultingpelletsin0.25xV2ofcoldPMbuffer,incubateatonicefor30minandcentrifugeagain(steps11-15).

18.	Theresultingcoldsupernatantis~90%puretubulinandiscalledtwice-cycledtubulin.Furtherpurificationisbyphosphocellulosecolumnchromatography.

19.	PrepareaPCcolumn(20mLPCresinin2.5cmdiametercolumn)andequilibratethecolumninPMbuffer.

20.	Priortocollection,1MMagnesiumSulfateisaddedtothecollectiontubestomakeafinalconcentrationof1mM.

21.

Applythetwice-cycledtubulintothePCcolumnandeludethecolumnwithPMbufferataflowrateof15mLperhourcollecting0.5-1mLfractions.Thefirsteluateispurifiedtubulinprotein.Donotmixwithlaterpeakfractions,whichcontainmicrotubule-associatedproteins(MAPs).Poolthepurifiedtubulin+/-MAPs,divideinto0.5mLaliquots,freezeinliquidnitrogenandstoreat-80°C.

Notes

1.	TubulinislABIleandyieldswillbegreatlyimprovedbyworkingrapidlyintheinitialstepsofthepreparation.