WholeCellExtracts REFERENCE:Hoffman,G.,Garrison,T.R.,andDohlman,H.G.,AnalysisofRGSproteinsinSaccharomycescerevisiae,MethodsEnzymol.344:617-631,2002. -Usesteriletechniqueandsterilesolutionsinsteps1to3.- 1.Usingasaturatedstarterculture,inoculate25to30mlofappropriatemediaina125mlflask. ***Sinceitisoftendifficulttoestimatethegrowthrateofyeast,itishelpfultostartseveral25mlcultures,eachwithadifferentdilutionofthestarterculture(e.g.1:100,1:300,1:900). 2.Growat30Cshaking(250rpm)untiltheOD600nm~1.0(Thisisusuallydoneovernight). ***Whengrowingseveralstrainsatonce,itislikelythattheywillallreachOD600nm~1.0atdifferenttimes.Ifdesired,sodiumazide(1Mstockinwater,dilutedtoafinalconcentrationof10mM)canbeaddedtoacultureonceitreachesanOD600nm~1.0.Theculturecanthenbeplacedoniceuntiltheothersareready. 3.Transfertoa50mlconicaltubeandcentrifugefor10minat2000xgat4C. 4.ResUSPendeachsamplein1mlof10mMsodiumazideandplaceonice. 5.CalculatethevolumeofresuspendedcellsthatwouldtranslatetoanOD600nmreADIngof10.Forexample,thiswouldequal1mlif10mlofcultureatOD600nm=1.0hadbeencentrifugedandresuspended. ***Thisstepisnecessarytoequalizetheamountofcells(andprotein)inagivenvolumeofwholecellextract. 6.Transferthecalculatedvolumeofresuspendedcellstoamicrofugetubeandcentrifugeat16,000xgfor1min. 7.Aspiratethesupernatent. 8.Resuspendthepelletin200ulof1XSDS-PAGEsamplebuffer. 9.Immediatelyplaceina100Cheatblockfor10min. 10.Allowthetubetocoolandadd200ulofglassbeads(Sigma,#G-8772). 11.Vortexathighspeedfor2min.Invertafterthefirstmin. ***Severaltubescanbevortexedatthesametimebyusingafoamtubefloatertoholdthemtogether. 12.Usinga21gaugeneedle,pokeaholeinthebottomofeachtubeandplaceitintoanewmicrofugetube. 13.Centrifugeat2000xgfor10sectoexpeltheliquidintothebottomtube,leavingtheglassbeadsinthetoptube. 14.Discardtheglassbeadsandcentrifugethebottomtubeat16,000xgfor2min.Thissedimentsanyinsolublematerial. 15.Transferthesupernatanttoanewmicrofugetube.Storeat-20C. 16.Whenreadytouse,heatat37Cfor10min,vortex,andcentrifugeat16,000xgfor1min. ***Keepinmindthatrepeatedfreezingandthawingcandegradetheproteinsample. 17.Immunoblotscanbeperformedusingstandardmethods. Updated01/23/02
