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Yeast Nuclear Extract (Small Scale) 组分提取 资讯
YeastNuclearExtract(SmallScale)
HahnLab(adaptedfromJ.Leatherwood,Ptashnelab)
lastmodified
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
BufferA | (650ml) |
18%Ficoll400(orpolysucrose400) | 117gFicoll |
10mMTris7.5 | 6.5ml1MTris7.5 |
20mMKAcetate | 13ml1MKAcetate |
5mMMgAcetate | 3.25ml1MMgAcetate |
1mMEDTA | 2.6ml0.25MEDTA |
0.5mMSpermidine | 82mgSpermidine |
0.15mMSpermine | 61microliters1.6MSpermine |
3mMDTTandproteaseinhibitorsareaddedjustpriortouse
TheFicolltakesmanyhourstodissolveandfrequentlyisstirredovernight.
BufferB | (250ml) |
100mMTrisacetatepH7.9 | 3gTris |
50mMpotassiumacetate | 12.5ml1MKAcetate |
10mMMgSO4 | 2.5ml1MMgSO4 |
20%glycerol | 50mlglycerol |
2mMEDTA | 2ml0.25MEDTA |
3mMDTTandproteaseinhibitorsaddedjustpriortouse
BufferC | (250ml) |
20mMHEPES7.6 | 1.19gHEPES |
10mMMgSO4 | 2.5ml1MMgSO4 |
1mMEGTA | 1ml0.25MEGTA |
20%glycerol | 50mlglycerol |
adjustpHwithKOH
3mMDTTandproteaseinhibitorsareaddedjustbeforeuse
BufferC+75mMAmmoniumsulfate | (1.5liters) |
20mMHEPES7.6 | 7.14gHEPES |
10mMMgSO4 | 15ml1MMgSO4 |
1mMEGTA | 6ml0.25MEGTA |
20%glycerol | 300mlglycerol |
75mMAmmoniumSulfate | 14.8gAmSO4 |
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
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