SUMEBBUFFER(1%SDS,8MUrea,10mMMOPS,pH6.8,10mMEDTA,0.01%bromophenolblue) TOMAKE100ml: 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.YeastCellLysates
ModifiedfromtheRineLab
Notethatproteaseinhibitorsareaddedfromstockstogive50folddilution.Example:20ulofstockper1mlbuffer.UseIMMEDIATELYafteraddingproteaseinhibitors.
ThisisusedforlysingyeastatpH6.8.ForpH8lyses,usethevariationofSUMEBwithTrisbufferknownasSUTEB.Also,bromophenolbluecanbeleftoutifdyeisnotdesired,suchasusingthelysateforimmunopreicipations.RapidProteinPrep
HorvathandRiezman,Yeast,1994
Stuffyouneed:SampleBuffer:0.06MTris-HCl,pH6.810%(v/v)glycerol2%(w/v)SDS5%(v/v)2-mercaptoethanol0.0025%(w/v)bromophenolblueGlassBeadPrep
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.