Weiss Walter, Görg Angelika
Technische Universität München, Fachgebiet Proteomik, Freising-Weihenstephan, Germany.
Methods Mol Biol. 2007;355:121-43. doi: 10.1385/1-59745-227-0:121.
Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteome analysis. 2-DE allows separation of highly complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance and delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or posttranslational modifications. 2-DE can resolve more than 5000 proteins simultaneously (approx 2000 proteins routinely) and can detect and quantify <1 ng of protein per spot. Today's 2-DE technology with IPGs has overcome the former limitations of carrier ampholyte-based 2-DE with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5 and 12 has allowed the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow pH range IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, permit the detection of low abundance proteins. In this article we provide a comprehensive protocol of the current 2-DE technology for plant proteome analysis and describe in detail the individual steps of this technique.
固定化pH梯度（IPG）二维凝胶电泳（2-DE）结合质谱（MS）进行蛋白质鉴定，是目前蛋白质组分析的主要方法。2-DE能够根据等电点（pI）、分子量（Mr）、溶解度和相对丰度分离高度复杂的蛋白质混合物，并生成完整蛋白质图谱，该图谱反映了蛋白质表达水平、异构体或翻译后修饰的变化。2-DE可同时分离5000多种蛋白质（常规约2000种蛋白质），每个斑点可检测和定量低至<1 ng的蛋白质。如今基于IPG的2-DE技术克服了以前基于载体两性电解质的2-DE在重现性、操作、分辨率以及极酸性和/或极碱性蛋白质分离方面的局限性。pH 2.5至12之间IPG的发展使得极碱性蛋白质的分析以及相应数据库的构建成为可能。窄pH范围的IPG提高了分辨率（ΔpI = 0.001），并与预分级方法相结合，能够检测低丰度蛋白质。在本文中，我们提供了用于植物蛋白质组分析的当前2-DE技术的综合方案，并详细描述了该技术的各个步骤。
