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1、蛋白质双向电泳总方案一、植物蛋白的提取1Chloroform/acetonemethod:ThismethodwasperformedaccordingtotheprotocoldescribedbyXieetal.(2007),withmodifications.Themodificationsineludeddifferenttissueamount,reagentvolume,andincubationtime.Thesemodificationsalsoappliedtotheotherthreemethodsdescribedbelow.About
2、0.5gofleafor1.5gofrootsamplewashomogenizedin4.0mLofextractionbuffer(0.1MKCI,0.5Mtris-baseatpH7.5,0.05MEDTA,and2%2-mercaptoethanol)for10min.Themixturewasshakenonicefor2handcentrifugedat12,000gfor15minat4°C・Thesupernatantwastransferredtoanewtube,andanequalvolumeofwatersaturatedchloro
3、formandisoamylalcohol(24/1,v/v)wasadded・Thetubewasshakenat4°Cfor30minandthenstoredonicefor10minbeforecentrifugationat10,000gfor10minat4°C.Theupperandbottomphaseswereremoved.Then4mLofwaterwasaddedtotheinterphasetogetherwithanequalvolumeofwater-saturatedchloroformandisoamylalcohol(24
4、/1,v/v);themixturewashomogenized,allowedtostandonicefor10min,andthencentrifugedat10,000gfor10minat4°C.Thetreatmentwasrepeatedtwice・Theinterphasewaswashedwithice-coldacetonethreetimes・Thefinalpelletswerevacuum-driedanddissolvedinresolubilizationsolution(8Murea,2Mthiourea,2%CHAPS,1%d
5、ithiothreitol[DTT],1%pharmalyte).2Phenol/ammoniumacetatemethod:ThismethodwasdevelopedfollowingtheprotocoldescribedbyHurkmanandTanaka(1986),withmod讦ication.About0.5gofleafor1.5gofrootsamplewashomogenizedin4.0mLextractionbuffer(50%phenol,0.45Msucrose,25mMEDTA,1%[v/v]2-mercaptoethanol
6、,250mMtris-base,pH8.8)for10min.Samplesweretransferredtophenol・resistantscrew-captubesandincubatedonamixerfor30minat4°Candthencentr讦ugedat5000gfor10minat4°C・Theupperphenol-phasewasremovedandaddedtofivevolumesofice-cold0.1Mammoniumacetateand1%2-mercaptoethanolin100%methanolandthenmix
7、edbeforeplacingat-20°Cfor2h.Precipitatedproteinwascollectedby10mincentrifugationat12,000g.Pelletswerethoroughlywashedtwicewith20mLof0.1Mammoniumacetatein100%methanol,followedbytwowasheswithice-cold80%acetone,andafinalwashwithice-cold70%ethanol.Thefinalpelletswerevacuum-driedanddiss
8、olvedinresolubilizationsol
