HorvathandRiezman,Yeast,1994;GottschlingLabSampleBuffer: 10ml 0.06MTris-HCl,pH6.8 0.6ml1MTris6.8 10%(v/v)glycerol 2ml50%glycerol 2%(w/v)SDS 2ml10%SDS 5%(v/v)2-mercaptoethanol 0.5ml2-mercaptoethanol 0.0025%(w/v)bromophenolblue 0.1mlSat.Bromphenolblue 4.9mlH2O
MakesampleBufferfreshbeforeuse.Canstorebufferfrozenat—20degreesfor~6months.
1.Growcellsovernight(~1x107cells/ml;A600=0.7)andcollect1.5mlcells(adjustvolumesaccordingtocelldensityofcultures)in1.5mlmicrofugetube(1minute,14000xg).Itisimportantnottogrowthecellstoahighdensityasthismethodwillnotworkwell. 10microlitersofasaturatedovernightcultureinYPDinnoculatedto5mlSD+essentialaminoacidsfor~16hrsgivesA600of0.5to1.0forwild-typecellsgrownat30degrees 150microlitersofYPDsaturatedculturedilutedto5mlYPDandgrownfor~5hrsat30degreesgivesanA600of~0.8forwild-typecells. 2.Washcells1Xwithwaterandcollectagainbycentrifugation. 3.ResUSPendcellsin100microliterssamplebuffer. 4.Heatat95degCfor5minutes. 5.Centrifuge14000xgfor5minutes.Load15microlitersperlaneonanSDSpolyacrylamidegel. Lastmodified