分蘖洋葱叶片总蛋白提取与双向电泳条件优化

Improvement of extraction method and two-dimensional electrophoresis conditions for proteome study on potato onion leaf

以分蘖洋葱叶片为试验材料,比较3种不同蛋白质提取方法对2-DE蛋白质双向电泳结果的影响,并对其中的蛋白上样量、SDS-PAGE凝胶浓度及双向电泳重复性进行筛选与优化。结果表明,与酚提取法和改良的酚提取法相比,TCA-丙酮提取法提取分蘖洋葱叶片总蛋白的操作简便,所得双向电泳图谱背景清晰,蛋白质点圆滑,数量及质量均较好,是一种切实可行的提取分蘖洋葱叶片总蛋白方法。应用Image Master 2D Platinum 7.0软件对图谱进行分析,凝胶上检测到超过650多个蛋白质点,不同凝胶之间蛋白质的匹配率达92.5%,具有较高的分辨率和重复性,建立一套适用于分蘖洋葱叶片总蛋白质组分析的双向电泳(2-DE)方法。

In this study, common potato onion leaf was taken as experimental material, leaf protein sample of young potato onion prepared by three methods. Two-dimensional electrophoresis was optimized in regard to protein extraction, the suitable concentration of protein per IPG gel, repetitions of experiment and SDS-PAGE parameters. The results showed that the protein yield extracted by TCA-acetone extraction method was much higher than that by phenolic extraction and modified phenolic method. The SDS-PAGE electrophoresis demonstrated that the protein isolated by TCA- acetone extraction method exhibited clearer bands and higher resolution than that by the other method with a fuzzy, low resolution. More than 650 proteins were detected in each gel and 92.5% of them were matched with each other in different gels stained by Coomassie Brilliant Blue analyzed by Image Master 2D Platinum 7.0 software, indicating the protocol with high repeatability and resolution. 2-DE （two-dimensional electrophoresis） protocol suited for the separation of proteins from potato onion leaf.