双向凝胶电泳比较三种常用蛋白质提取方法

组织(或细胞)的蛋白质提取效率直接影响蛋白质双向凝胶电泳(2-DE)的分辨率.为探索建立适用于人乳腺癌细胞株MCF-7蛋白质提取的最佳条件,比较目前在双向凝胶电泳中常用的3种蛋白质提取方法对MCF-7细胞总蛋白的提取效率.MCF-7细胞经培养后,分别采用M-PER试剂、标准裂解液或含硫脲裂解液提取其总蛋白质,然后进行双向凝胶电泳,并根据凝胶上蛋白质斑点的丰度和分布特点判断所得双向电泳图谱的质量,以确定MCF-7细胞蛋白质提取的相对最佳方法.结果显示,M-PER试剂法得到的图谱分辨率较低,蛋白质主要集中分布在分子量15~70kD,pH4.7~6.3的范围内;标准裂解液法得到的图谱分辨率有所提高,蛋白质分布比M-PER试剂法得到的图谱广;硫脲裂解液法得到的图谱是三者中分辨率最高的,尤其是高丰度蛋白和高分子量蛋白分离效果比前两者好.结果表明,在3种常用的蛋白质提取方法中,硫脲裂解液对细胞蛋白质的溶解性最佳,相对更适合于提取MCF-7细胞的蛋白质,并与双向凝胶电泳条件更兼容.

Comparison of Three Protein Extraction Methods by Two-Dimensional Electrophoresis

Protein extraction from tissue or cells is a key step to achieve high-resolution protein separation in two dimensional electrophoresis (2-DE). Three routine cellular total protein extraction methods were compared in order to determine an optimal one for human breast cancer cell line MCF-7. The cultured MCF-7 cells were lysed by M-PER kit, standard lysis buffer or improved lysis buffer, respectively. Then the extracted total proteins were subjected to 2-DE, and the best extraction method was determined by the indexes of protein distribution and abundance on corresponding silver-stained gel. Data showed that use of M-PER kit gave the lowest resolution, in which most proteins were distributed in the pI ranging from 4.7 to 6.3 with molecular weight between 15 kD and 70 kD. Standard lysis buffer improved protein resolution with broader protein distribution pattern. Improved lysis buffer generated the best resolution among these three methods, especially for the high-abundance and high molecular weight proteins. Based on above results, we concluded that the improved lysis buffer has the best protein solubilization ability, which renders it much more suitable for cellular protein extraction from MCF-7, and is more compatible with the conditions of 2-DE.