大型海藻龙须菜蛋白提取及双向电泳体系的优化

Optimization of Extraction Method and Two Dimensional Electrophoresis Conditions for Proteome of Macroalgae Gracilaria Lemaneiformis

本文以龙须菜为材料,比较了尿素/硫脲法,改良丙酮沉淀法,Trizol法3种不同蛋白质提取方法;并从胶条长度及pH范围,上样量,分离胶浓度等因素探讨了双向电泳技术体系的优化.结果表明:采用Trizol法辅以超声破碎提取龙须菜总蛋白效果优于尿素/硫脲法和改良丙酮沉淀法;同时,采用pH 4-7,11 cm胶条,上样量为800μg,分离胶浓度为12%的双向电泳条件可获得背景清晰,分辨率好的二维电泳图谱.该体系也可以为龙须菜属和江蓠属其他海藻的蛋白质双向电泳分析提供参考.

In order to establish a two-dimensional polyareylamide electrophoresis(2DE) system for proteomic study of G. lemaneiformis, three different protein extraction methods (sodium phosphate buffer grinding method,TCA/acetone precipitation and Trizol method)were compared. Electrophoresis conditions were optimized, including the length of IPG strip,loading quantity,the concentration of SDS-PAGE gel. The result showed that Trizol method can display much more protein spots than the other two methods. Based on the Trizol extracting method, lower backgrounds and higher resolution 2-DE image were obtained using 800μg protein loading quantity;11 cm, pH 4-7IPG strip;12%SDS-PAGE gel. A suitable two dimensional electrophoresis system was established for proteomic analysis of G. lemaneiformis and other macroalgae.