An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE
An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE
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通过 2-DE 溶解、还原和转移人乳腺癌膜富集组分的优化程序
DOI：
10.1002/elps.200700035
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发表时间：
2007-09-01
期刊：
影响因子：
2.9
通讯作者：
Wan, Mingxi
中科院分区：
文献类型：
--
作者：
Ruan, Yusong;Wan, Mingxi
尽管基于双向电泳的膜蛋白质组学研究取得了一定的成果，但膜蛋白质组学中的整体膜蛋白和外周膜蛋白的分离仍然是一个挑战。以人乳腺癌细胞系MCF-7为材料，研究了Tris、还原剂、杯装量和SDS对膜蛋白溶解和2-DE分离的影响。向样品溶液中添加三羟甲基氨基甲烷改善了膜富集级分的溶解，并且在20 mM三羟甲基氨基甲烷下获得了最佳质量的凝胶图案。三丁基膦（TBP），一种还原剂，在2-DE过程中，是不是最佳的，因为它不仅减少了疏水蛋白的溶解，但也导致一些蛋白质，如热休克蛋白60，prohibitin，和肌动蛋白，被解析成一串点。TBP与DTT联合应用可提高二维超声图像的分辨率。杯加载显着促进了膜蛋白进入IPG条带，并可视化了超过1000个高分辨率的蛋白点。采用这种策略，通过在IEF中间加入DTT，ATP合酶α链首次在2-DE凝胶图中被分解为两个相邻的点。平衡缓冲液中的高SDS浓度增强了转移，并增加了凝胶中约50%蛋白质斑点的染色强度，但也导致一些斑点的损失。
The separation of integral and peripheral membrane proteins is still a challenge, although many achievements have been made in the 2-DE-based membrane proteomics. Using a human breast cancer cell line, MCF-7, we investigated the influences of Tris, reducing reagents, cup loading, and SDS on membrane protein solubilization and separation by 2-DE. The addition of Tris to the sample solution improved the solubilization of the membrane-enriched fraction, and the best-quality gel patterns were obtained at 20 mM Tris. Tributylphosphine (TBP), a reducing agent, was not optimum in the 2-DE process because it not only decreased the solubilization of hydrophobic proteins but also caused some proteins, such as hsp60, prohibitin, and actin, to be resolved to a string of spots. However, when combined with DTT, TBP could improve the resolution of 2-DE patterns. Cup loading significantly facilitated the entrance of membrane proteins into IPG strips and over 1000 protein spots with high resolution were visualized. Adopting this strategy, an ATP synthase alpha chain was resolved into two adjacent spots for the first time in 2-DE gel patterns through the adding DTT in the middle of the IEF. A high SDS concentration in the equilibration buffer enhanced the transfer and increased the staining intensity of similar to 50% of the protein spots in the gels, but also resulted in losses of some spots.