一种适用于双向电泳分析的高效真菌线粒体提取及蛋白质制样方法

An Efficient Method of Isolation and Protein Preparation of Fungal Mitochondria for Two-dimentional Electophoresis

用液氮冷冻研磨法破碎真菌菌丝细胞,通过差速和蔗糖密度梯度离心分离纯化板栗疫病菌线粒体,所得线粒体产率(质量比)约为1/10~4。电子显微镜观察和蛋白印迹表明,所制备的线粒体完整性好,没有检测到其它细胞成分的污染。使用膜蛋白裂解液溶解线粒体制备蛋白样品,将蛋白样品200μg上样于pH3~10,24cm的非线性胶条进行等电聚焦,电聚焦后再进行第二向SDS-PAGE电泳分离,经银染获得重复性好、背景清晰、分辨率高(680±15个蛋白质点)的凝胶图谱。随机选择10个蛋白质点进行质谱分析,9个获得有效注释,均为线粒体特异性蛋白质,表明所制备的蛋白样品非常适合双向电泳分析及其后续的质谱鉴定。

Mitochondrial sample of chestnut blight fungus （Chryphonectria parasitica） was prepared by liquid nitrogen grinding and purified with a combination of differential and sucrose density gradient centrifugation. The mitochondrial yield by this method was about 1/104 （w/w）. The integrity and purity of the mitochondrial preparation were confirmed by electron microscopy and Western blot analysis. The mitochondrial protein samples were prepared by dissolving purified mitochondria into membrane lysis buffer. An amount of 200 p^g mitochondrial proteins was loaded onto a pH 3~10, 24 cm NL IPG strip for isoelectrofocusing and then separated in a PAGE, which resulted in a 2D map with good reproducibility, clear background and high resolution （680＋ 15 protein spots） by silver staining. Nine of the ten randomly picked protein spots were effectively identified and they all were mitochondrion-specific. The experimental results demonstrated that the mitochondrial protein samples were suitable for 2-DE and MS analysis.