Grow100mlyeastcellsindesiredmediaovernighttoanA600of~1.0(0.6to1.2workswell).Forgrowthinminimalmedia,1mlofasaturatedovernightcultureinminimalmedia(Syntheticdextrose(SD)withonlytherequiredaminoacids)wasinoculatedto100mlofthesamemediaandgrown~16hrsat30degrees.Harvestcellsandwashwith20mlofcoldextractionbufferina50mltube.ResUSPendcellsin0.5mlextractionbuffercontainingDTTandproteaseinhibitorsinamicrocentrifugetubewithalockingtop(marshtube).Add~500microlitersofglassbeads.Inthecoldroom,shaketubesonthefoamringofthevortexmixerplatformattopspeedfor1min.Transfertoicefor1min.Ihavedoneupto20extractsatonce.Repeatforatotalof10minofvortexing.Brieflymicrocentrifugetoremoveallliquidandleavebehindmostoftheglassbeads.Centrifugeattopspeedat4degreesfor15minandremovesupernatant,beingcarefultoavoidanyglassbeads.AssayproteinconcentrationusingBioRadorPierceassays.Freezeextractsandstoreat-80deg.Expect10-15mg/mlprotein.ExtractBuffer:100mMTrispH7.9250mMAmmoniumSulfate1mMEDTA10%GlycerolBeforeuse,addDTTto0.5mM(lowconcentrationsoIPreactionscanbedonedirectly)And1Xproteaseinhibitorsfromthefollowingstocksolutions:0.1MPMSF(100x)16mg/mlEthanol;Storeat-20degreesBenzamidine(100X);31mg/mlH2O;Storefrozenat-20degreesLeupeptin(500X);0.15mg/mlEthanol;Storeat-70degreesforlessthan6monthsPepstatin(200X);0.28mg/mlmethanol;Storeat-20degrees.Chymostatin(2,500X);5mg/mlDMSO;Storefrozenat-20degrees