Comparison of protein extraction methods from Brassica napus roots and optimization of two-dimensional electrophoresis | 甘蓝型油菜根系可溶性蛋白的提取及双向电泳体系的优化
2011
Wang Zhenhua, Huazhong Agricultural University,Wuhan(China) | Wang Zhifang, Huazhong Agricultural University,Wuhan(China) | Chen Shuisen, Huazhong Agricultural University,Wuhan(China)
中国人. 摘 要：以甘蓝型油菜品种青油10号幼苗期根系为材料,采用三氯乙酸/丙酮沉淀法、酚提法及酚提取-甲醇/醋酸铵沉淀法分别提取甘蓝型油菜根系可溶性蛋白。以牛血清蛋白为标样,使用Bradford法测定蛋白质浓度,计算蛋白提取率,并对所提取的蛋白样品进行聚丙烯酰胺凝胶电泳（SDS-PAGE）及双向电泳（2-DE）检测,所得2-DE凝胶图片经PD Quest软件分析,分别检测得到644、645、669个蛋白点。对双向电泳条件进行优化,选择改进的酚提法提取蛋白,采用17 cm（pH 5~8）的IPG胶条、蛋白上样量150μg及10%的SDS-PAGE胶含量,可以获得分辨率高、重现性高的2-DE凝胶图片。
显示更多 [+] 显示较少 [-]英语. The aim of the study is to establish a procedure on root protein extraction applied for proteomics analysis in Brassica napus seedlings.Three methods（TCA/acetone,phenol extraction and phenol extraction-methanol/ammonium acetate precipitation） for protein extraction were assessed with Brassica napus cultivar ＂Qingyou 10＂ root at seedling stage as experiment materials.The protein concentration was determined using Bradford protein assay with BSA as a standard,and the protein samples were separated by SDS-PAGE and two-dimensional electrophoresis（2-DE） in turn.Then,the 2-DE gel images were analyzed by PD quest,detecting 644,645,669 clear spots,respectively.Evaluating comprehensively the efficiency of protein extraction among the three methods and optimal 2-DE condition,modified phenol extraction,along with the 2-DE parameters,17 cm（pH 5-8） IPG strip,150 μg protein loading volume and 10% PAGE gel concentration,were chosen to obtain high reproducible and clear 2-DE gel image,which could satisfy the demand for protein isolation and identification in proteomics study.
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