蛋白双向电泳总方案-管胶.doc

蛋白双向电泳总方案-管胶.doc蛋白质双向电泳总方案一、植物蛋白的提取Chloroform/acetonemethod:ordingtotheprotocoldescribedbyXieetal.(2007),,reagentvolume,(,-,,and2%2-mercaptoethanol),000gfor15minat4°,andanequalvolumeofwatersaturatedchloroformandisoamylalcohol(24/1,v/v)°Cfor30minandthenstoredonicefor10minbeforecentrifugationat10,000gfor10minat4°-saturatedchloroformandisoamylalcohol(24/1,v/v);themixturewashomogenized,allowedtostandonicefor10min,andthencentrifugedat10,000gfor10minat4°--driedanddissolvedinresolubilizationsolution(8Murea,2Mthiourea,2%CHAPS,1%dithiothreitol[DTT],1%pharmalyte).Phenol/ammoniumacetatemethod:ThismethodwasdevelopedfollowingtheprotocoldescribedbyHurkmanandTanaka(1986),(50%phenol,,25mMEDTA,1%[v/v]2-mercaptoethanol,250mMtris-base,)for10m