Steinberger Birgit, Mayrhofer Corina
Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria.
Methods Mol Biol. 2015;1295:305-21. doi: 10.1007/978-1-4939-2550-6_23.
Methods for analyzing the phosphorylation status of proteins are essential to investigate in detail key cellular processes, including signal transduction and cell metabolism. The transience of this post-translational modification and the generally low abundance of phosphoproteins require specific enrichment and/or detection steps prior to analysis. Here, we describe three gel-based approaches for the analysis of differentially expressed phosphoproteins. These approaches comprise (1) the sequential fluorescence staining of two-dimensional (2-D) gels using Pro-Q(®) Diamond and SYPRO(®) Ruby dyes to visualize and quantify phosphoproteins in total cellular lysates as well as (2) affinity enrichment of phosphoproteins in conjunction with sequential fluorescence staining of the 2-D gels and (3) affinity enrichment of proteins prior to pre-electrophoretic fluorescence labeling and 2-D gel electrophoresis.
分析蛋白质磷酸化状态的方法对于详细研究关键细胞过程（包括信号转导和细胞代谢）至关重要。这种翻译后修饰的短暂性以及磷酸化蛋白通常较低的丰度要求在分析之前进行特定的富集和/或检测步骤。在此，我们描述三种基于凝胶的方法用于分析差异表达的磷酸化蛋白。这些方法包括：（1）使用Pro-Q® Diamond和SYPRO® Ruby染料对二维（2-D）凝胶进行顺序荧光染色，以可视化和定量总细胞裂解物中的磷酸化蛋白，以及（2）磷酸化蛋白的亲和富集结合2-D凝胶的顺序荧光染色，和（3）在预电泳荧光标记和2-D凝胶电泳之前进行蛋白质的亲和富集。
