Very alkaline immobilized pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins
Very alkaline immobilized pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins
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DOI：
10.1002/elps.1150180306
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发表时间：
1997-03-01
期刊：
影响因子：
2.9
通讯作者：
Madjar, JJ
中科院分区：
文献类型：
--
作者：
Gorg, A;Obermaier, C;Madjar, JJ
通常因载体两性电解质聚焦的阴极漂移而丢失的碱性蛋白质，或通过 NEPHGE 分离而重现性有限的碱性蛋白质，可以通过使用先前公布的方案（Gorg 等人，Electrophoresis 1988, 9, 531-546）的固定 pH 梯度 (IPG) 4-10 和 6-10，在平衡条件下通过二维 (2-D) 电泳得到很好的分离。在本研究中，我们将 pH 梯度扩展到 pH 12 和 IPG 8-12、9-12 和 10-12，以分析非常碱性的蛋白质。为了从 HeLa 细胞和小鼠肝脏中获得高度可重复的核糖体蛋白二维模式，需要在 pH 工程、凝胶组成和运行条件方面采取不同的优化步骤，例如用二甲基丙烯酰胺替代丙烯酰胺，以及向 IPG 再脱水溶液中添加或不添加甲基纤维素的异丙醇（以抑制反向电渗流）。来自鸡红细胞核的组蛋白以及红细胞的总细胞提取物也在稳态条件下成功分离。由于 IPG 9-12 中等电聚焦的选择性，其中酸性较强的蛋白质会放弃凝胶，因此可以省略组蛋白提取之前繁琐的细胞核制备过程。
Basic proteins normally lost by the cathodic drift of carrier ampholyte focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two-dimensional (2-D) electrophoresis under equilibrium conditions using immobilized pH gradients (IPGs) 4-10 and 6-10 using a previously published protocol (Gorg et al., Electrophoresis 1988, 9, 531-546). In the present study we have extended the pH gradient to pH 12 and IPGs 8-12, 9-12 and 10-12 for the analysis of very basic proteins. Different optimization steps with respect to pH engineering, gel composition and running conditions, such as substitution of acrylamide by dimethylacrylamide and addition of iso-propanol with and without methylcellulose to the IPG redydration solution (in order to suppress the reverse electroosmotic flow) were necessary to obtain highly reproducible 2-D patterns of ribosomal proteins from HeLa cells and mouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under steady-state conditions. Due to the selectivity of isoelectric focusing in IPG 9-12, where the more acidic proteins abandon the gel, the tedious procedure of nuclei preparation prior to histone extraction can be omitted.