-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.