Mayer Kristin, Albrecht Sally, Schaller Andreas
Institute of Plant Physiology and Biotechnology, University of Hohenheim, Emil-Wolff-Str. 25, Stuttgart, 70599, Germany,
Methods Mol Biol. 2015;1306:167-76. doi: 10.1007/978-1-4939-2648-0_13.
Two-dimensional (2D) gel electrophoresis combines isoelectric focusing in the first and SDS polyacrylamide gel electrophoresis in the second dimension to separate complex mixtures of proteins with unequalled resolution and sensitivity. It is well suited for the analysis of posttranslational protein modifications as most of them affect the isoelectric point and, therefore, the focusing behavior of the protein in the first dimension. It is particularly useful for low-abundance proteins, as it provides a first indication of PTMs, before establishing methods for protein isolation. For targeted proteomics of more abundant proteins, 2D electrophoresis itself may be the method of choice for the isolation of posttranslationally modified isoforms of the protein of interest for mass spectrometric analyses. Protein phosphorylation can be detected by use of phospho-specific stains or antibodies, or by comparing spot patterns of a protein sample before and after phosphatase treatment. Here we describe a simple method, combining 2D gel electrophoresis and western blot analysis with dephosphorylation by λ-phosphatase to analyze the phosphorylation status of oxophytodienoic acid reductase 3 in protein extracts from different organs of tomato and Arabidopsis plants.
二维（2D）凝胶电泳将第一向的等电聚焦与第二向的SDS聚丙烯酰胺凝胶电泳相结合，以无与伦比的分辨率和灵敏度分离复杂的蛋白质混合物。它非常适合分析蛋白质的翻译后修饰，因为大多数修饰会影响等电点，进而影响蛋白质在第一向的聚焦行为。它对低丰度蛋白质特别有用，因为在建立蛋白质分离方法之前，它能提供翻译后修饰的初步指示。对于更丰富蛋白质的靶向蛋白质组学，二维电泳本身可能是分离感兴趣蛋白质的翻译后修饰异构体以进行质谱分析的首选方法。蛋白质磷酸化可以通过使用磷酸特异性染色剂或抗体，或者通过比较蛋白质样品在磷酸酶处理前后的斑点模式来检测。在这里，我们描述了一种简单的方法，将二维凝胶电泳和蛋白质印迹分析与λ-磷酸酶去磷酸化相结合，以分析番茄和拟南芥植物不同器官的蛋白质提取物中氧植物二烯酸还原酶3的磷酸化状态。
