Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis.
Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis.
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DOI：
10.1186/1477-5956-3-5
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发表时间：
2005-06-07
期刊：
影响因子：
2
通讯作者：
Coorssen JR
中科院分区：
文献类型：
--
作者：
Churchward MA;Butt RH;Lang JC;Hsu KK;Coorssen JR
由于溶解性问题，双向凝胶电泳对疏水膜蛋白的分析一直受到固有困难概念的阻碍。我们通过改变增溶缓冲液的洗涤剂成分和各种市售的非离子和两性离子洗涤剂以及类洗涤剂磷脂来优化提取方案。在用SDS-PAGE进行初步分析后，对人红细胞膜、小鼠肝膜和小鼠脑膜进行定量二维分析，用含有两性离子洗涤剂MEGA 10（癸烷酰- n -甲基葡萄糖酰胺）和两性离子脂质LPC （1-lauroyl溶血磷脂酰胆碱）的缓冲液提取，结果显示，与常用的2-DE洗涤剂CHAPS（3[（3-胆酰胺丙基）二甲酰胺]-1-丙磺酸）的提取相比，选择性提高。三种去污剂的混合物在斑点数、密度和分辨率上都有明显的改善。在脑膜蛋白质组的分析中观察到实质性的改进。本研究表明，优化的洗涤剂组合，加上严格的样品处理和电泳方案，可以使用二维电泳简单有效地分析膜蛋白质组。
The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids. After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed. This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.