烟草根系蛋白质双向电泳样品制备方法的比较

Comparison of Protein Extraction Methods for Two-dimensional Electrophoresis in Tobacco Root

为探索适于烟草根系蛋白质组学研究的样品制备方法,以栽培品种K326生长期根系为材料,比较了常规裂解液法、TCA/丙酮沉淀法、Trizol抽提法和酚法等4种总蛋白质的提取方法。对制备的样品进行聚丙烯酰胺凝胶电泳(SDS-PAGE)及双向电泳(2-DE)检测。结果表明:酚法最佳,所获蛋白在单向SDS-PAGE中形成条带数目多而清晰;经双向电泳分离用银染显色,可检测约548个蛋白点,图谱分辨率较好,蛋白点清晰,主要集中在等电点pH5～8、相对分子量25.0～70.0kD,可有效解析烟草根系的蛋白质组。

In order to develop an efficient protein extraction method suitable for tobacco proteome analysis,four protocols for total protein extraction in tobacco root,lysis-buffer method,TCA/acetone precipitation method,Trizol extraction method and phenol method were assessed with tobacco cultivar K326 root at growth stage as experiment materials.The protein samples were separated by SDS-PAGE and two-dimensional electrophoresis（2-DE）.It was found that the phenol method was the best one resulting in the most protein bands and the best resolution in SDS-PAGE.About 548 clear protein spots were detected by 2-DE and silver staining with isoelectric points at pH 5～8,and relative molecular weight of protein ranged from 25.0 kD to 70.0 kD.