心肌线粒体双向凝胶电泳技术分析

目的：建立并优化心肌线粒体蛋白质组研究的双向凝胶电泳（two-dimensional gel electrophoresis，2-DE）技术。方法：分离200g雄性SD大鼠心肌线粒体，抽提心肌线粒体的全蛋白，以固相pH梯度、等电聚焦为第一向和垂直SDS聚丙烯酰胺凝胶电泳为第二向行双向凝胶电泳，对样品制备、上样量、电泳参数、不同固定方式等相关技术进行优化。凝胶行硝酸银染色，扫描仪获取凝胶图像并用PDQuest软件进行分析。结果：45％乙醇和5％乙酸的固定液对蛋白质固定效果最好；构建了心肌线粒体高分辨率和重复性的双向凝胶电泳图谱。结论：建立了SD大鼠心肌线粒体蛋白质组的双向凝胶电泳分析技术。

Two-dimensional Gel Electrophoresis Technical Analysis of Mitochondrial Proteins from the Heart of Rats

Objective:To establish and optimize two-dimensional gel electrophoresis technique for proteomics of mitochondrial proteins from heart of rats. Methods:Mitochondria were isolated from the hearts of 200g male SD rats. Total proteins extracted from mitochondria were applied to the two-dimensional gel electrophoresis. Immobiline pH gradients isoelectric focusing was used as 1D,vertical SDS-PAGE as 2D. Sample separation, protein loading,electrophoresis parameters and fixation styles were optimized. The gels were stained by silver, scanned by ImageScanner, analyzed with PDQuest software. Results: 45% alcohol and 5% acetic acid was found to have the best protein fixation effect. The 2-DE electrophoretogram with high resolution and reproducibility of heart mitochondrial proteins have been successfully established Conclusion: The 2-DE technique for proteomics of mitochondrial proteins from heart of SD rats have been established