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

关键词:  心肌蛋白  载体两性电解质  双向凝胶电泳

Key words:  proteins of cardiac muscle  carrierampholytes  two-dimensional electrophoresis

摘要:

［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pH 3~10和pH 4~6 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，pH 3~10和pH 4~6 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。

Abstract:

［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. ［Conclusions］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.