YeastNuclearExtract(SmallScale)

HahnLab(adaptedfromJ.Leatherwood,Ptashnelab)

lastmodifiedMon,Aug27,2001

CellGrowth

3litersofcellsaregrowninYPD(3%glucose)toA600of3-5.AntifoamA(twodropsfromaP200pipetman)isaddedtomediabeforeautoclaving.

Forwildtypecells,~2.5mlofYPDovernightinoculatedperliterat5:30pmgivesA600of~3-4at9:00amifcellsaregrownat30o.Forwild-typecellsgrownat25o,10-12mlinoculatedperliterworksbest.Slowgrowingmutantsneedanywherebetween10-120ml/literinoculateddependingonthestrain.

Extractpreparation

Day1

HarvestCellsin1literbottles(4Krpmfor10min.).DrainexcessmediaaswellaspossIBLeandweighcells.Expectedyieldis20-35gcells.Anythinglessthan18gwillgivepoorextracts.Ifcellsareovergrown,lyticasewillworkpoorlyinspheroplastingcells.

ResUSPendcellpelletsin35ml50mMTris7.5,30mMDTT.Usuallythiscanbedonebygentlyshakingthecentrifugebottles.Leavecellsin1literbottles.Incubateat30ofor15min.

Pelletcells(4Krpmfor8min.)andresuspendin20mlYPD/S.Add15ml2Msorbitol.Add15mlrecombinantlyticase.Incubateat30owithoccasionalgentlemixing.

Alternative:Insteadofrecombinantlyticase,canalsouseZymolyase100TfromICN.Use12-18mgperprep.Theamountrequiredcanvaryfrom~12-18mgdependingontheyeaststrain(often,wild-typetakesthehigheramount).Ifusingzymolyase,addinoneextraYPD/Swashingstepat4degrees.

Checkprogressofspheroplastingevery15min.Tocheck,mix4microlitersofcellswith4microliters1%SDSonaglassslide.Observethenumberofcellghostsundermicroscope.Incubatecellsuntilabout80%spheroplastsareobtained.Thiscantakeanywherefrom30min.to21/2hours.Ifcellsarespheroplastingslowlyafter1hr,anextra5-10mloflyticasecanbeadded.However,ifcellswereovergrown(A600>5),thecellsmayneverspheroplast.Spheroplastingisalsosomewhatstraindependent.

Afterspheroplastinghasreachedabout80%,add100mlYPD/S(roomtemp)andpelletcells(4Krpmfor12min).

Resuspendcellsin250mlYPD/S(roomtemp)andincubateat30degreesfor30min.toallowcellstorecover.Theresuspensionofspheroplastsworksbestifasmallvolume(~50ml)ofYPD/Sisfirstaddedandcellsareresuspendedusingabakingspatula.ThenaddtheremainingYPD/S.

Pelletcells(4Krpmfor12min.)andresuspendin200mlcoldYPD/S(4degrees).Resuspendasinthepreviousstep.Keepeverythingcoldfromthispointon.Cellscanbekeptoniceforanhourorsoifothercellsarestillspheroplasting.

(addanextraYPD/Swashifusingzymolyaseinsteadoflyticase)

Pelletcells(4Krpmfor12min)andresuspendin250mlcold1Msorbitol.

Pelletcells(4Krpmfor12min)andanddrainsorbitolmediaaswellaspossible(careful-sometimesthespheroplastpelletisnotverytight).Resuspendin100mlBufferAat4ocontainingDTTandproteaseinhibitors.Transferto250mlbeaker.

Passcellsuspension1XthroughYamamotoLH1homogenizerat500rpmincoldroom.TransferhomogenizedcellstoGSAbottles.

Note:WehavealsohadsuccessdouncingthespheroplaststhreetimesinaBdounceifthehomogenizerisnotavailable,butthehomogenizerisrecommended

Spin5Krpmfor8min.TransfersupernatanttonewGSAbottles.Donotworryabouttheslimyloosepelletthatalsotransfers.Repeat.

Spinsupernatant5Krpmfor5min.TransfersupernatanttonewGSAbottle.Repeat.Bythelast(fourth)spin,theslimynonpelletedmaterialshouldbenearlygoneandthepelletsfirm.

Optional:Ifthesupernatantcontainsaverylargeamountofslimymaterialafterthislastspin,onemorespinat5Kfor5mincanbedone.However,donotdoanyadditionallowspeedspinsafterthis.

Transfersupernatantto50mlcentrifugetubesandpelletcrudenuclei.Spin13Krpmfor30min.inSS34rotor.Removesupernatantbydumpinganddiscard.Drainpellets.

Resuspendcrudenuclearpelletswithasmallspatulain10mlBufferBandtransferto50mlscrewcaptubes.Theprepcanbestoppedatthispoint.Quickfreezeandstoreresuspendednuclearpelletsat-70degrees.

Day2

Thawnucleioniceandmeasurevolume.Add3Mammoniumsulfate(pH7.5)to0.5Mfinalconcentration(1/5originalvolumeofnuclei)andimmediatelymixandincubateonrollerincoldroomfor30min.After10min,breakupanylumpswithaglassrod.Thissteplysesnuclei.

TransfertoSW28thickwalledultracentrifugetubesandspinat28Krpmfor90min.at4o.

Carefullyremovesupernatantwith5mlPipette(andpasteurpipetteifnecessary)beingcarefultoavoidthepellet.Donotworryaboutthewhitefloatingmaterial.Transferto50mlscrewcaptube.

Add0.35gsolidammoniumsulfate/mlsupernatantandimmediatelyincubateoncoldroomrollerfor30min.Theammoniumsulfatecanbeaddedallatonceifanumberofprepsarebeingdone.However,itisbestifammoniumsulfateisaddedslowlywhilestirringsupernatantinabeaker.ThepHshouldremainabove7(italmostalwaysdoes)butshouldbechecked.AdjustpHwith1MNaOHifnecessary.

TransfertothickwalledultracentrifugetubesandspininSW28at10Krpmfor20minat4o.Removesupernatantbydumpingandrespinpelletsat10Krpmfor4min.Carefullyremoveallremainingsupernatantwithapasteurpipette.

ResuspendpelletsinBufferC(+0salt)containingDTTandproteaseinhibitors.Dependingonproteinpelletsize,resuspendin1.5-0.4mlbuffer.Thiscanbedonewithasmalldouncehomogenizerorabluepipettetipdependingontheamountofprotein.

DialyzenuclearextractvsBufferC+75mMammoniumsulfateat4o.Dialyzevs500mlbufferwith3changesofbufferover4.5hourstotal.

Aliquotextractandstoreat-70degrees.Extractsshouldbe25-50mg/mlinprotein.

BioRadProteinAssayofextracts.

ExtractsaresometimesdifficulttogetareproduciblemeasurementofproteinconcentrationusingtheBioRadassay.Thismodifiedmethodworkswell.

Diluteextract1/4in0.1%SDS.Add1-2microlitersofdilutedextractto0.8mlH20ina13x100mmdisposabletesttube.Add1microliter0.1%SDStoproteinstandards.Add0.2mldyereagent.After10min,readabsorbanceatA595.

Buffersandsolutionsfornuclearextracts(volumesgivenarefor62lextracts)

50mMTris,7.5250ml

1.5gTrisin250mlH2O

pHto7.5withHCl

Beforeuse,add4.6mgDTT/ml

YPD/S2.5litersatroomtemp.(YPDwith1Msorbitol)

25gYeastextract

50gBactopeptone

50gDextrose(glucose)

455gSorbitol

H2Oto2.5literstotal

YPD/S1.5liters(4degrees)

15gYeastextract

30gBactopeptone

30gDextrose

273gSorbitol

AddH2Oto1.5liters

1MSorbitol(4degrees)

273gSorbitol

H2Oto1.5liters

3mMDTTandproteaseinhibitorsareaddedjustpriortouse

TheFicolltakesmanyhourstodissolveandfrequentlyisstirredovernight.

3mMDTTandproteaseinhibitorsaddedjustpriortouse

adjustpHwithKOH

3mMDTTandproteaseinhibitorsareaddedjustbeforeuse

adjustpHto7.6withKOH

ProteaseInhibitorsandDTTareaddedjustbeforeuse(0.46gBenzamidine,0.69gDTT,15mlPMSF,3ml500XLeupeptin,7.5ml200Xpepstatin,0.6ml2,500Xchymostatin).

Zymolyase100T(ICN)

Thisisreportedlycontaminatedwithproteases,soextracareisneededtowashspheroplasts.Dissolveat6mg/mlin50mMTriswith2Xconcentratedproteaseinhibitors.Incubate10minonicebeforeusing.Thismaterialdoesnotdisolvewell,sokeepinsuspensionaswellaspossible.

ProteaseinhibitorsandDTT

0.1MPMSF(100x)

16mg/mlEthanol

Storeat-20degrees

0.2MDTT

32mg/mlH2O

Storefrozenat-20degrees

Benzamidine(100X)

31mg/mlH2O.

Storefrozenat-20degrees

Leupeptin(500X)

0.15mg/mlEthanol.

Storeat-70degreesforlessthan6months

Pepstatin(200X)

0.28mg/mlmethanol

Storeat-20degrees.

Chymostatin(2,500X)

5mg/mlDMSO

Storefrozenat-20degrees