几种不同提取方法对Hela细胞总蛋白双向电泳结果的影响  

Comparative 2-DE Analysis of Protein Expression Profiles in Hela Cells with Different Extraction Protocols

作　　者：孙丽翠[1] 胡家[2] 郑君芳[1] 苏雷[3] 贺俊崎[1] 

机构地区：[1]首都医科大学生物化学与分子生物学系 [2]首都医科大学蛋白质组学检测中心 [3]首都医科大学宣武医院胸外科

出　　处：《首都医科大学学报》2008年第4期451-454,共4页Journal of Capital Medical University

基　　金：国家自然科学基金(30572183,30772573);教育部博士点基金(20060025011 );教育部新世纪优秀人才支持计划(NCET-06-0184);北京市教育委员会科技发展计划重点项目(KZ200610025013);首都医科大学基础临床合作基金(2007JL13)资助项目~~

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。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 freezlng-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.

关 键 词：双向电泳 蛋白表达谱 蛋白提取 

分 类 号：Q503[生物学—生物化学]