【摘要】 为建立一套适合海滨木槿叶片蛋白质组学分析的双向电泳体系,以海滨木槿无性系叶片为材料,分别采用裂解液法、三氯乙酸-丙酮沉淀法、改良酚抽法分离纯化蛋白质,选用24 cm pH 3～10的IPG胶条,并对不同上样量、等电聚焦程序、平衡时间等电泳条件进行优化。结果表明:采用改良酚抽法提取蛋白质,上样量为每IPG胶条1 000μg,总聚焦功率90 kV.h,平衡时间15 min,胶体考马斯亮蓝染色,可得到蛋白质点数目多、分辨率高的双向电泳图谱。重复性试验结果显示,该体系具有很好的稳定性及可重复性,可满足海滨木槿蛋白质组学研究的要求。更多还原

【Abstract】 To establish a two-dimensional polyacrylamide gel electrophoresis(2-D PAGE) system for leaf proteomic analysis of Hibiscus hamabo,three methods including lysis-buffer method,TCA-acetone precipitation method,and the improved phenol extraction method were applied for leaf protein isolation and purification,and the key technologies of electrophoresis including the sample loading quantity,isoelectric focusing conditions,equilibrium time were optimized by taking 24 cm pH 3-10 immobilized pH gradient(IPG) strips.The results indicated that the reproducible 2-D gel with more spots and high resolution electrophoresis diagram could be obtained by using the improved phenol extraction method,with 1 000 μg of loading protein sample per IPG strip,90 kV·h isoelectric focusing(IEF) time,15 min of equilibrium time and staining with colloidal coomassie brilliant blue as the most suitable treatment conditions.The results of repetitive experiments showed that this electrophoresis system was stable and reproducible,which could meet the demand for the proteomics research on H.hamabo.更多还原