【摘要】 目的比较几种不同的提取方法对Hela细胞总蛋白双向电泳图谱的影响。方法采用反复冻融裂解法、超声加冰浴裂解法、室温振摇及试剂盒纯化法提取Hela细胞总蛋白,进行双向凝胶电泳(2-DE)。以7cmpH3-10NL固相pH梯度胶条做一向等电聚焦,上样量为300μg,SDS-PAGE做二向垂直电泳,改进的胶体考马斯亮蓝染色,ImageMaster2D Platinum软件分析电泳图谱。结果反复冻融裂解法得到的2-DE图谱条纹很多,蛋白点数较少(400±);超声加冰浴裂解和室温振摇法得到的2-DE图谱横向条纹明显减少,蛋白点数明显增加(分别为700±和800±);进一步用试剂盒纯化法得到的2-DE图谱基本上没有蛋白拖尾现象,且加Destreak试剂后蛋白点数有所增加(由800±→900±),但纯化的同时有低丰度蛋白的丢失。结论超声加冰浴裂解和室温振摇法处理细胞,可得到条纹较少、蛋白点数较多较全的2-DE图谱;进一步用蛋白纯化试剂盒和Destreak试剂纯化样品能更好地解决碱性端的横向拖尾,得到较多的蛋白点。更多还原

【Abstract】 Objective The aim of this study is to compare protein expression profiles in two-dimensional electrophoresis analysis using different protein extraction protocols.Methods Total proteins were extracted from Hela cells with freezing-thaw,ultrasonic treatment,gentle shake treatment and 2-D clean up kit respectively.Samples were separated by two-dimensional electrophoresis,in which 300 μg of protein samples were applied to 7cm pH 3-10 NL immobilized pH gradient gel strips.Protein spots were visualized by modified colloidal CCB staining and then analyzed with Image Master 2D Platinum Software.Results Samples from freezing-thaw treatment exhibit fewer protein spots(400±) and more horizontal stripes on 2-DE maps.In contrast,more spots(700± and 800±) and fewer horizontal stripes were detected with ultrasonic or gentle shake treatment,and stripes were further reduced with 2-D clean up kit.However,2-D clean up kit detected less protein spots especially for low-abundance proteins,and addition of Destreak reagent to 2-D clean up kit can increase the efficiency of protein yield on the 2-D image(800±→900±).Conclusion Protein expression profiles of Hela cells in 2-DE analysis were obtained with few stripes and more protein spots after ultrasonic or gentle shake treatment to extract proteins.Combination of 2-D clean up kit with Destreak reagent for protein extraction can diminish horizontal stripes,especially in the alkaline end.更多还原