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以生长到Feekes 8.5时期小麦旗叶为试验材料,通过差速离心结合两相法提取并纯化质膜蛋白,进而在裂解液选择、SDS-PAGE胶浓度及蛋白质上样量等方面对质膜蛋白质双向电泳体系进行了优化.结果表明,采用6.4%PEG 3 350/Dextran T-500(W/W)两相体系可以获得纯度高达87.9%质膜微囊.经TCA-丙酮法裂解蛋白,以12%SDS-PAGE分离胶对900μg质膜蛋白进行双向电泳,在2-DE图谱上可分辨出173个蛋白点.建立了一套用于小麦旗叶高纯度质膜的提取方法及其蛋白质组学双向电泳体系.
The flag leaf of wheat growing to the period of Feekes 8.5 was used as the experimental material, and the plasma membrane protein was extracted and purified by differential centrifugation combined with two-phase method. Then, the plasma membrane protein was bi-directionally expressed in the aspects of lysis solution selection, SDS-PAGE gel concentration and protein loading The results showed that the microcapsules with purity of up to 87.9% could be obtained by the two-phase system of 6.4% PEG3 350 / Dextran T-500 (W / W) .The protein was cleaved by TCA- SDS-PAGE separation gel electrophoresis of 900μg plasma membrane protein, in the 2-DE map can be identified 173 protein spots.A set of a method for the extraction of wheat flag leaf high purity plasma membrane and its proteomics two-dimensional electrophoresis system.