Asano Tomoya, Nishiuchi Takumi
Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, 920-0934 Kanazawa, Japan.
Methods Mol Biol. 2011;744:225-33. doi: 10.1007/978-1-61779-123-9_16.
Two-dimensional gel electrophoresis (2-DE) has often been used to compare protein expression pattern between different samples. This method is also useful to compare protein expression between wild-type and RNAi plants. 2D-DIGE (difference gel electrophoresis) was developed to perform quantitative proteomics of two or more samples. In this method, proteins are fluorescently labeled by Cy2, Cy3, or Cy5 and are mixed and subjected to electrophoresis on the same gel, thereby gel-to-gel variations are eliminated. We perform phosphoproteomics between Arabidopsis wild-type and a stress-activated MAPK (mitogen-activated protein kinase) mutant after the phytotoxin treatment. For this purpose, proteins are extracted from both samples, and then phosphorylated proteins are purified by phosphoprotein enrichment columns. Purified proteins are fluorescently labeled by different CyDyes using the minimum labeling method. The labeled protein samples are separated on the same gel by 2-DE, and gels are scanned by a variable mode laser imager. Then, high-resolution images are analyzed by 2D analysis software. Thus, we identified several phosphoprotein spots that were differentially accumulated between wild-type and mapk mutant.
二维凝胶电泳（2-DE）常被用于比较不同样品之间的蛋白质表达模式。该方法对于比较野生型植物和RNA干扰植物之间的蛋白质表达也很有用。差异凝胶电泳（2D-DIGE）技术的开发是为了对两个或更多样品进行定量蛋白质组学分析。在此方法中，蛋白质用Cy2、Cy3或Cy5进行荧光标记，然后混合并在同一凝胶上进行电泳，从而消除了凝胶间的差异。我们在植物毒素处理后对拟南芥野生型和应激激活的丝裂原活化蛋白激酶（MAPK）突变体进行磷酸化蛋白质组学分析。为此，从两个样品中提取蛋白质，然后通过磷酸化蛋白质富集柱纯化磷酸化蛋白质。使用最小标记法用不同的CyDye对纯化的蛋白质进行荧光标记。标记的蛋白质样品通过2-DE在同一凝胶上分离，并用可变模式激光成像仪扫描凝胶。然后，用二维分析软件分析高分辨率图像。因此，我们鉴定出了几个在野生型和mapk突变体之间差异积累的磷酸化蛋白质斑点。
