Cells:

WegrowourCHOswithMEM[[alpha]](withoutnucleosides)+10%BovineCalfSerumandpenn/strep/glutamine.Foraprepitisbesttogrowtwentylargeplates(150mM)andthecellsshouldbegrowntooverconfluence-tilltheystartpilinguponeachother(youwillneedtofeedthemfrequentlytokeepthemhappy)

ProtocolRationale:

TheprotocolisidenticaltoTim"spublishedinVol134ofMethodsinEnzymology.ThekeystepisthelysiswhichsolubilizescentrosomesawayfromnucleibyverylowionicstrengthlysisaftertreatmentofcellswithnocodazoleandcytochalasinB.ThereleasedcentrosomesarethencentrifugedontoaFicollcushion(toavoidpelleting)andtheinterfacebetweenthelysateandtheFicolliscollectedandthecentrosomesareconcentratedonasucrosegrADIent.FractionsareassayedbyspindownanddoubleIFwith5051serumandanti-tubulinandthepooledfractionsarefrozeninliqN2.

Whatyouneed:

Equipment:

• refractometer
• SorvallwithcoldHB-4
• UltrawithcoldSW27
• Somesortofgradientfractionatorwithafractioncollector
• 1630mlcorextubes(cold)

Solutions:

Alotofthesesolutionsarew/w;Timsaysthattomaketheseweighoutsucroseandthenaddbuffertillweightis100g.

Wash&Lysis:

PE:10mMPIPES,1mMEDTA,8mMBME

Makea50XstockandpHto7.2withKOH

LB:1mMTris-HCl,8mMBME

MakeTrisas2MstockandpHto8.0withHCl

LB+0.5%NP-40:

(warmfor30"to37deg.CtoensureNP-40hasdissolved)

PBS:130mMNaCl,2mMKCl.8mMNa2HPO4,2mMKH2PO4

Makea10Xstock

Nightbefore:

Make600mlof

• 1XPBS
• 0.1XPBS
• 0.1XPBS,8%(w/w)ultrapuresucrose
• 8%(w/w)ultrapuresucrose
• LB(w/oBME):add280ulBMEbeforeuse
• LB+0.5%NP-40(w/oBME):add280ulBMEbeforeuse

andputallthesebuffersincoldroom

SucroseGradients:

Useultrapuresucrose

20%(w/w)and62.5%(w/w)sucrosein1xPE+0.1%TX-100.Make100gramsofeachasfollows:

Weighoutsucroseandthenonthebalanceadd1XPE+0.1%TX-100tillweightis100grams.

Justbeforepouringgradientsadd28ulBME/50g

Pourgradientsnightbeforeorduringdrugtreatment(seelater).Ifinditeasiesttopournightbeforeandstoreincold.

Topourgradients-firstputa5mlheavysucrosepadandthenpourgradientontopofthat.FortheprepoutlinedbelowIpour2gradientsinSW27tubes:

4mlheavysucrosepad(use62.5%orhigher).

16mlgradient.

Thecentrosomesareveryclosetothebottomofthisgradient.Thepadeliminatesthemfromenteringthecurveofthetubeandalsogivesalittleleewayinsettingupthefractionation.

Ficollcushion:

20%(w/w)Ficoll(MW400,000)in1XPE+0.1%NP-40

1. MakeupPE+0.1%NP-40(noBME).
2. Weighout10gFicoll.
3. AddPE+0.1%NP-40tilltotalweight=50grams.
4. StiratRTforseveralhourstodissolveandstorecold.
5. Beforeuse,add28ulBME

Protocol:

1. Warmup300mlofCHOmediumto37deg.CandaddcytochalasinB(150ulof10mg/ml)andnocodazole(300ulof10mg/ml)
2. Makesureallbuffersareincoldroom,thereisrockerinthecoldroomandthereisagoodaspiratorinthecoldroom.HookupasawedoffpipettotheaspiratorandmakesurethereisaLARGEtrap(atleast4L)
3. Addmediumwithdrugsto10platesofcells.Addmediumwithdrugstotheother10plates45"later.
4. After90"indrugmediumprocessfirst10plates:bringtocoldroomandwashwiththefollowingbuffers:
   1XPBS
   0.1XPBS,8%(w/w)sucrose
   8%(w/w)sucrose
   LB

   andthenpipeton

   LB+0.5%NP-40(10mls/plate)

   (Thesewashesmustbedonerapidly-allwashesshouldbeunder1"perplate.ThewayIdothemistopouronthewashbufferfromabeakerorgradcylinder(~30mls),immediatelyrockbackandforthandaspirateASAPbeforepouringonthenextwash.Itiscriticaltodothisquicklytogetgoodlysisoryouwilllosemostofthecentrosomesinthenuclearpellet).AfteraddingtheLB+0.5%NP-40transfertheplateontoarockerinthecoldroom.

5. After10"pipetoffthelysateintoa30mlcorextube.Add1/50volof50XPE(0.5mlfor25mls;withBME)
6. SpintubesinHB-4for3"at3000rpmat4deg.C
7. Transfersupetoafreshcorextubeandunderlaywith2mlofFicollcushion(Iusea6ccsyringewith16gaugeneedlewithathinpieceoftygontubingwhichIcanslidetothebottomofthetube).
8. Spinat12,700rpmfor15at4deg.Cfor15".Assoonasspinisstarted,processthenextsetofplatesthroughsteps4-7.
9. Aspiratesupetillapprox.2mlabovecushionandthencollectinterfacewithapasteur-seeTim"sprotocol.Collect~2ml/tubeandpool.CheckFicollconcentrationbyrefractometryanddiluteto10%(w/w)orlower-necessarytomakesureitlayersontothesucrosegradientanddoesn"tsink.
10. Finishcollectinginterfacesfromsecondsetofplatesandthenpoolallcollectedinterfaces,ensureFicollis<10%(w/w)andloadonto1gradient.Spin1hr-1hr30",SW27at2deg.C.
11. Fractionategradientfrombottom-0.3-0.5mlfractionsandreadsucroseconcentrationbyrefractometry.Assayfractionsbetween48and60%(w/w)sucrose.5uloffraction+5mlPE-mixwellandpelletontocoverslips:12,500rpmfor15"at4deg.CinHB-4.Postfixinmethanol(-20deg.C)for5"andrehydrateanddo5051+antitubulinfollowedbyanti-mouse,anti-humansecondaries.Assesspeakbyconcentrationofdoublestainingdots,poolandfreezeinliqN2in10ulaliquots.

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