Solutions:

BufferA(HypotonicBuffer):1L10mMHEPESpH7.910ml1MHEPES,pH7.91.5mMMgCl21.5ml1MMgCl210mMKCl3.33ml3MKCl0.5mMDTT500µl1MDTT0.2mMPMSF1ml0.2MPMSF

BufferB(S100extractionbuffer):500ml0.3MHEPESpH7.9150ml1MHEPES,pH7.91.4MKCl233.3ml3MKCl30mMMgCl215ml1MMgCl2LowSaltBuffer:1L20mMHEPESpH7.920ml1MHEPES,pH7.925%Glycerol250mlGlycerol20mMKCl6.67ml3MKCl1.5mMMgCl21.5ml1MMgCl20.2mMEDTA400µl0.5MEDTA0.5mMDTT500µl1MDTT0.2mMPMSF1ml0.2MPMSF

HighSaltBuffer:1L20mMHEPESpH7.920ml1MHEPES,pH7.925%Glycerol250mlGlycerol1.2MKCl400ml3MKCl1.5mMMgCl21.5ml1MMgCl20.2mMEDTA400µl0.5MEDTA0.5mMDTT500µl1MDTT0.2mMPMSF1ml0.2MPMSF

BufferD(DialysisBuffer):For2L20mMHEPES,pH7.91MHEPES7.940ml20%GlycerolGlycerol400ml100mMKCl3MKCl66.67ml0.2mMEDTA0.5MEDTA800µl0.5mMDTT1MDTT1ml0.2mMPMSF0.2MPMSF2ml

SplicingExtractPreparation

1.ResUSPendcellpelletin~5packedcellvolumesofcoldPBS.Splitintoadditional250mlconicaltubesifnecessary.Spinat1500rpmfor10minutesinJ-6.

2.Resuspendthewashedcellpelletin5packedcellvolumesBufferA(HypotonicBuffer).Spinat1500rpmfor5minutesinJ-6.

3.AddBufferAuptoafinalof3originalpackedcellvolumes(i.e.,add~2volumes[donotexceedatotalof3volumes]).Incubateonicefor10minutes.

4.TransferthecellstoaWheatonADouncehomogenizerandlysewith~10strokes.StainanaliquotwithTrypanBlueandcheckfor>90%lysisonmicrosope.

5.Spinat2000rpmfor15minutesinJ-6topelletnuclei.

6.SavethesupernatantforS100extract(seebelow).

7.Transferthenuclearpellettoaglassbeakercontainingastirbarandadd1/2packednuclearvolumeofLowSaltBuffer.

8.Whilestirringgently,add1/2packednuclearvolumeofHighSaltBuffer(1.2MKCl).HomogenizeinDounceifnecessary.

9.Stirfor30minutesincoldroom.

10.Transferthenuclearextractto30mlCorextubesandspinat10,000rpmfor30minutesinHB-4rotorat4°C.

11.Removethesupernatantanddialyzefor~2hoursin>50volumesBufferDincoldroom.Changebufferanddialyzeagainst>50volumesfor~2.5hours.

12.Transferto30mlCorextubesandspinat10,000rpmfor20-30minutesinHB-4rotorat4°C.

13.Aliquotin1mlaliquotsandfreezeinliquidnitrogen.Storeat-80°C.

S100extract

A.Mixcytoplasmicextractwith0.11volumesbufferB.

B.Spinat39,400for60minutesinOakridgetubesinTi70rotorat4°C.

C.Dialyzefor~2hoursthen~2.5hoursin>50volumesBufferDlackingPMSFincoldroom.

D.Spin14,000rpmfor30minutesinSS-34rotorinOakridgetubes.

E.AliquotS100into1mlfractionsandfreezeonN2andstoreat-80°C.