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Yeast Cell Lysates
YeastCellLysates
ModifiedfromtheRineLab
- Growcellsto0.3to0.5O.D.(600)(~1E7cells/ml).Useabout1.0to5.0ODofcellsperlysate;ifyourcellsareat0.5ODperml,use2mltogiveyou1.0ODofcellsinyourlysate.Use2.0mlEppendorftubesastheflatbottomsmakeglassbeadlysiseasier.
- Harvestcellsbyspinninginmicro-centrifugefor2minutes,decantsupernatant.
- Add200ulofSUMEBbuffer+proteaseinhibitors.
- Add100ulof0.5mmAcidWashedGlassBeads.
- Vortexinmultivortexerorbyhand,3X1minspeedsetting7onourcurrentmachine.
- Incubatefor10minat65°Corwhatevertemperatureisdesired.
- RemovethelysatefromthebeadswithabluePipettetiptoanew1.5mleppendorftube.
- Spin5minutestoclarify.Removesupernatanttoanew1.5mleppendorftube.
- Usesupernatantdirectlytoloadgelordotblot.
SUMEBBUFFER(1%SDS,8MUrea,10mMMOPS,pH6.8,10mMEDTA,0.01%bromophenolblue)
TOMAKE100ml:
- 1.0ml1MMOPS,pH6.8stocksolution
- 1.0gSDS,or10ml10%SDSstocksolution
- 48.05gUrea
- 2.0ml0.5MEDTAstocksolution
- 1.0ml1%bromophenolblue
50XSTOCKProteaseInhibitors(storeat-20°C)PMSF(87mg/ml)([500mM]phenylmethylsulfonylfluoride)
TOMAKE30ml:Dissolve2.61gPMSFinDMSOtoequalafinalvolumeof30ml.
LEUPEPTINANDPEPSTATIN(5mg/ml)
TOMAKE10ml:Dissolve50mgLEUPEPTINorPEPSTATINinDMSOtoequalafinalvolumeof10ml.
TPCK(5mg/ml)(tosylphenylalaninechloromethylketone)
TOMAKE10ml:Dissolve50mgTPCKinDMSOtoequalafinalvolumeof10ml.*Note:TheEDTAintheSUMEbufferisalsoaproteaseinhibitor.
RapidProteinPrep
HorvathandRiezman,Yeast,1994
Stuffyouneed:SampleBuffer:0.06MTris-HCl,pH6.810%(v/v)glycerol2%(w/v)SDS5%(v/v)2-mercaptoethanol0.0025%(w/v)bromophenolblue- Growcellsovernight(~1E7cells/ml)andcollect1.5mlcellsin1.5mlmicrofugetube(1minute,14000xg).
- Washcells1Xwithwaterandcollectagainbycentrifugation.
- ResUSPendcellsin100µlsamplebuffer.
- Heatat95degCfor5minutes.
- Centrifuge14000xgfor5minutes.
- Load~25µlperlaneonanSDSpolyacrylamidegel.
GlassBeadPrep
Stuffyouneed:SampleBuffer:0.06MTris-HCl,pH6.810%(v/v)glycerol2%(w/v)SDS5%(v/v)2-mercaptoethanol0.0025%(w/v)bromophenolblue0.1MPMSF0.5MBenzamidine1.Grow25mlofcellstomid-log.2.Spindown(2500rpmfor5minutes).Wash1Xwithwaterandspinagain.3.Resuspendin1mlofwaterandtransferto1.5mlmicrofugetube.Spindownfor5secondsandpouroffwater.4.Resuspendin0.5mloficecoldSampleBufferwithfreshlyaddedPMSF(0.5mM)andbenzamidine(0.5mM).5.Addglassbeads(~0.5ml).6.Vortexonhigh4Xfor45secondswith30secondsoniceinbetweeneachmixing.7.Spinfor5minutesinmicrofugeat4degC.8.Transfersupernatanttoanewtubeandboilfor5minutes.9.Load10-15µlonaproteingel.
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