小麦旗叶高纯度质膜的提取及蛋白质组学双向电泳体系的建立

Preparation of Plasma Membrane with High Purity from Wheat Flag Leaves for 2-DE Proteomics

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.

A purified plasma membrane fraction was isolated using differential centrifugation combined with aqueous two-phase partitioning from wheat Feekes 8.5 flag leaves. The lysis buffer was optimized for two-dimensional electrophoresis （2-DE） analysis, and the conditions of different gel concentrations and sample loading were compared. The results showed that high purity plasma membrane of 89.2% was obtained at 6.4% PEG 3350/Dextran T-500 （W/W） in our two-phase system. The plasma membrane proteins were extracted by the TCA/acetone method, loaded in the amount of 900 μg and resolved by a 17 cm pH 4 - 7 IPG and 12% SDS-PAGE gel, then visualized by CBB G-250 staining. A total of 173 protein spots were detected by the PDQuest 2DE 8.0. 1 software. The optimization of 2-DE protocol allowed a suitable condition for proteomic analysis of plasma membrane from wheat flag leaves.