甜瓜幼苗根系总蛋白提取方法的筛选及其电泳条件的建立

Extraction of total protein and establishment of electrophoresis system of muskmelon root

以‘西域1号’甜瓜苗期根系为材料,分别采用改进的TCA/丙酮沉淀法、Tris/酚提取-甲醇/醋酸铵沉淀法及SDS/酚提取法提取总蛋白,进行SDS电泳和双向电泳分离,扫描后的胶图利用Image Master 2D Elite 6.01软件进行分析,重点分析胶图总点数、匹配点数和差异蛋白点数。结果表明:Tris/酚提取-甲醇/醋酸铵沉淀法得到的总蛋白含量最高;TCA/丙酮沉淀法分离得到的蛋白条带和蛋白点数最少,且对分子量较高的偏碱性区域的蛋白提取效果差,与其他2种方法相比明显丢失了较多的蛋白。因此,进行甜瓜幼苗根系总蛋白质组分析时,可采用后2种方法,其中Tris/酚抽提法更简单易行。

Three improved methods, TCA/acetone precipitation, Tris/phenol extraction-metha- nol/ ammonium acetate precipitation and SDS/phenol extraction, were adopted to extract root proteins of muskmelon ＇Xiyu1＇. The prepared samples were isolated by one-dimensional SDS electrophoresis and two-dimensional electrophoresis respectively, then total spots, matched spots and differential protein spots in scanning copy of gels were ana Elite 6.01. The results showed that Tris/phenol extraction-methano Y / zed by Image Master 2D ammonium acetate pre cipitation received the highest protein content among the three methods, while TCA/acetone precipitation got the minimum spots and lost much of basic proteins. It is suggested that the second and the third methods could be used for root proteome analysis of muskmelon seedlings, and Tris/phenol extraction methanol is more available for its easier operation.