YeastCellLysates

ModifiedfromtheRineLab

1. Growcellsto0.3to0.5O.D.(600)(~1E7cells/ml).Useabout1.0to5.0ODofcellsperlysate;ifyourcellsareat0.5ODperml,use2mltogiveyou1.0ODofcellsinyourlysate.Use2.0mlEppendorftubesastheflatbottomsmakeglassbeadlysiseasier.
2. Harvestcellsbyspinninginmicro-centrifugefor2minutes,decantsupernatant.
3. Add200ulofSUMEBbuffer+proteaseinhibitors.
4. Add100ulof0.5mmAcidWashedGlassBeads.
5. Vortexinmultivortexerorbyhand,3X1minspeedsetting7onourcurrentmachine.
6. Incubatefor10minat65°Corwhatevertemperatureisdesired.
7. RemovethelysatefromthebeadswithabluePipettetiptoanew1.5mleppendorftube.
8. Spin5minutestoclarify.Removesupernatanttoanew1.5mleppendorftube.
9. Usesupernatantdirectlytoloadgelordotblot.

Notethatproteaseinhibitorsareaddedfromstockstogive50folddilution.Example:20ulofstockper1mlbuffer.UseIMMEDIATELYafteraddingproteaseinhibitors.

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

ThisisusedforlysingyeastatpH6.8.ForpH8lyses,usethevariationofSUMEBwithTrisbufferknownasSUTEB.Also,bromophenolbluecanbeleftoutifdyeisnotdesired,suchasusingthelysateforimmunopreicipations.

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.

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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

1. Growcellsovernight(~1E7cells/ml)andcollect1.5mlcellsin1.5mlmicrofugetube(1minute,14000xg).
2. Washcells1Xwithwaterandcollectagainbycentrifugation.
3. ResUSPendcellsin100µlsamplebuffer.
4. Heatat95degCfor5minutes.
5. Centrifuge14000xgfor5minutes.
6. Load~25µlperlaneonanSDSpolyacrylamidegel.

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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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