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.