大鼠心肌蛋白双向凝胶电泳技术的建立

Establishment of a two-dimensional electrophoresis technology on proteins of cardiac muscle

[目的]建立大鼠心肌蛋白质组研究的双向凝胶电泳（two-dimensional gel electrophoresis,2-DE）技术。[方法]提取SD大鼠心肌总蛋白,以载体两性电解质pH梯度等电聚焦为第一向电泳,垂直SDS-聚丙烯酰胺凝胶电泳为第二向电泳,完成2-DE,考马斯亮蓝染色后扫描获取凝胶图像并用PDQuest软件进行分析。对2-DE中关键环节,如调节pH3~10和pH4~6载体两性电解质的比例、样品离心处理、增加等电聚焦电压和离子强度等方面进行优化。[结果]通过样品处理后离心除去多糖成分,pH3~10和pH4~6载体两性电解质的比例调至1∶2,改变电泳条件,成功消除横向拖尾、分辨率较高的心肌蛋白双向电泳图谱。每张图谱可检测到超过150个蛋白质点,匹配率大于55%。[结论]建立了SD大鼠心肌蛋白的双向凝胶电泳分析技术,该方法可行、重复性好,所提供的方案具有一定参考意义。

[Objective] Establishes the technology of two-dimensional gel eleetrophoresis on rat cardiac muscle protein group studies（2-DE）.[Methods] The total proteins of cardiac muscle tissues were separated by carrierampholytes pH gradients based gel electrophoresis for first and the vertical SDS-polyacrylamide gel electrophoresis for second to the line of bidirectional gel electrophoresis two-dimensional gel electrophoresis（2-DE）.The gels were dye by the Coomassie brilliant blue stained,and scanned to gain gel image and use PDQuest for gel image analysis.Regulate proportion of ampholyte pH 3~10 and pH 4~6 and centrifuge sample,Conditions of the two-dimensional gel electrophoresis have been optimized.[Results] After the optimization（the proportion of ampholyte pH 3~10 and pH 4~6 was regulated into 1/2,sample was centrifuged to remove polysaccharide and so on.） the horizontal trailing protein spots was decreased and the resolution of those were improved.Over 150 protein spots were detected.[Conclusion] The two-dimensional gel electrophoresis analysis of cardiac muscle protein was successfully established.The method is rapid and has good reproducibility.The result has definite referenced vale.