Protein extraction from yeast(酵母蛋白质提取)

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1、Proteinextractionfromyeast1.GlassbeadslysisConzelmannA,RiezmanH,DespondsC,BronC.1988.Amajor125kdmembraneglycoproteinofSaccharomycescerevisiaeisattachedtothelipidbilayerthroughaninositol-containingphospholipid.EMBOJ7:2233±2240.2.rapidproteinextractioninoptimizedSDSsamplebufferHorwathA,R

2、iezmanH.1994.RapidproteinextractionfromSaccharomycescerevisiae.Yeast10:1305±1310.SampleBuffer:10ml0.06MTris-HCl,pH6.8 0.6ml1MTris6.810%(v/v)glycerol 2ml50%glycerol2%(w/v)SDS 2ml10%SDS5%(v/v)2-mercaptoethanol 0.5ml2-mercaptoethanol0.0025%(w/v)bromophenolblue 0.1mlSat.Bromphenolblue  4.9

3、mlH2OMakesampleBufferfreshbeforeuse.Canstorebufferfrozenat—20degreesfor~6months.1.Growcellsovernight(~1x107cells/ml;A600=0.7)andcollect1.5mlcells(adjustvolumesaccordingtocelldensityofcultures)in1.5mlmicrofugetube(1minute,14000xg).Itisimportantnottogrowthecellstoahighdensity.10microlite

4、rsofasaturatedovernightcultureinYPDinnoculatedto5mlSD+essentialaminoacidsfor~16hrsgivesA600of0.5to1.0forwild-typecellsgrownat30degrees150microlitersofYPDsaturatedculturedilutedto5mlYPDandgrownfor~5hrsat30degreesgivesanA600of~0.8forwild-typecells.2.Washcells1Xwithwaterandcollectagainbyc

5、entrifugation.3.Resuspendcellsin100microliterssamplebuffer.4.Heatat95degCfor5minutes.5.Centrifuge14000xgfor5minutes.Load15microlitersperlaneonanSDSPAGE3.post-alkalineextractionVitalyV.Kushnirov2000.Rapidandreliableproteinextractionfromyeast.Yeast2000;16:857±860.about2.5OD600(whichconst

6、itutesabout2.3mgofwetweight)ofyeastcellswereharvestedbycentrifugationfromliquidcultureorscrapedofftheagarplateusingabacteriologicalloop.Thesecellswereresuspendedin100mldistilledwater,added100ml0.2MNaOH,incubatedfor5minatroomtemperature,pelleted,resuspendedin50mlSDSsamplebuffer,boiledfo

7、r3minandpelletedagain.About6μlsupernatantwastypicallyloadedperlaneofmini-gel(Bio-RadMini-Proteancell).Thesamplebuffer(0.06MTris±HCl,pH6.8,5%glycerol,2%SDS,4%bmercaptoethanol,0.0025%bromophenolblue)wasslightlymodi®edfromstandard(Laemmli,1970).SDS-Boil-BeadsWholeCellExtractsREFERENCE:H