Görg Angelika, Lück Carsten, Weiss Walter
Technical University of Munich, Munich, Germany.
Methods Mol Biol. 2008;424:277-86. doi: 10.1007/978-1-60327-064-9_22.
Prefractionation procedures not only aid in reducing sample complexity, but also permit loading of higher protein amounts within the separation range applied to two-dimensional electrophoresis (2-DE) gels and thus facilitate the detection of less abundant protein species. Hence we developed a simple, cheap, and fast prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated Sephadex gels, containing chaotropes, zwitterionic detergents and carrier ampholytes. After IEF, up to ten Sephadex gel fractions alongside the pH gradient are obtained, and then applied directly onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of 2-DE. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and full compatibility with subsequent 2-DE, because the prefractionated samples have not to be eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with IEF in IPG strips. This sample prefractionation method has been successfully applied for the separation, detection and identification of low abundance proteins from pro- and eukaryotic samples.
预分级程序不仅有助于降低样品复杂性，还能在二维电泳（2-DE）凝胶的应用分离范围内加载更高的蛋白量，从而便于检测丰度较低的蛋白质种类。因此，我们基于在含有离液剂、两性离子去污剂和载体两性电解质的粒状葡聚糖凝胶中进行的平板等电聚焦（IEF），开发了一种简单、廉价且快速的预分级程序。IEF后，可沿着pH梯度获得多达十个葡聚糖凝胶级分，然后直接将其应用于相应窄pH范围固定化pH梯度（IPG）条带的表面，作为2-DE的第一向。该技术的主要优点是预分级的蛋白质能高效地从葡聚糖IEF级分电泳转移到IPG条带中，无需任何样品稀释，并且与后续的2-DE完全兼容，因为预分级的样品无需洗脱、浓缩或脱盐，葡聚糖级分中的载体两性电解质的量也不会干扰IPG条带中的IEF。这种样品预分级方法已成功应用于从原核和真核样品中分离、检测和鉴定低丰度蛋白质。
