SteveHahn

LastmodifiedFri,Oct16,1998

Beforetryingthismethod,tryrapidproteinprepwhichinvolvesboilingyeastinSDSPAGEbuffer

1.Grow100mlyeasttoA600~1.0.Iftheyeastareovergrown,useproportionallylesscellsfortheprep.2.Collectcellsbycentrifugationandwashin20mlcoldextractionbuffer(containingDTTandproteaseinhibitors).Keepeverythingcoldfromthispointon.3.ResUSPendcellsin600microlitersextractionbufferandtransferto13x100mmglasstesttubesonice.Add400microlitersglassbeads.4.Inthecoldroom,vortexfor1minoniceandkeeponiceforatleast1minwhilevortexingothertubes.5.Repeatthevortexingstep(step4)foratotalof5times.6.Spincellsinprecooledclinicalcentrifugefor3minattopspeed.7.Usinga1mlpipetman,removethesupernatant,leavingmostoftheglassbeadsbehind(transferringsomeoftheyeastisOK).Transfertoamicrocentrifugetube.8.Inthecoldroom,spinattopspeedinthemicrofugefor30min.Removesupernatanttoanewtubebeingcarefulnottocarryoveranycelldebris.9.MeasureproteinconcentrationusingBioRadassayandfreezeinaliquots.Load20-40microgramsonproteingelforwesternanalysis.

ExtractionBuffer:200mMTrispH8.0150mMAmmoniumsulfate10%glycerol1mMEDTAAddfreshondayofextractpreparation:2mMDTTPMSFBenzamidineLeupeptinPepstatinChymostatinProteaseinhibitors0.1MPMSF(100x)16mg/mlEthanolStoreat-20degrees0.2MDTT(100x)32mg/mlH2OStorefrozenat-20degreesBenzamidine(100X)31mg/mlH2O.Storefrozenat-20degreesLeupeptin(500X)0.15mg/mlEthanol.Storeat-70degreesforlessthan6monthsPepstatin(200X)0.28mg/mlmethanolStoreat-20degrees.Chymostatin(2,500X)5mg/mlDMSOStorefrozenat-20degrees