Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE.
Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE.
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DOI：
10.1016/j.mex.2015.11.007
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发表时间：
2015
期刊：
影响因子：
1.9
通讯作者：
Sakamoto S
中科院分区：
文献类型：
--
作者：
Sugiyama Y;Katayama S;Kameshita I;Morisawa K;Higuchi T;Todaka H;Kinoshita E;Kinoshita-Kikuta E;Koike T;Taniguchi T;Sakamoto S
多PK抗体识别各种物种中的多种蛋白激酶。使用Phos标签的SDS-PAGE，磷酸化的蛋白质与相应的非磷酸化的蛋白质相比被可视化为较慢的迁移带。这种组合方法可用于检测各种细胞内蛋白激酶的表达和磷酸化状态的变化。蛋白激酶的表达和活性通过调节磷酸化信号在多种细胞功能中发挥重要作用。检测蛋白激酶最常用的工具是蛋白激酶特异性抗体，磷酸化位点特异性抗体用于检测活化的蛋白激酶。使用这些抗体，一次仅分析一种激酶，然而，用于分析细胞中一组蛋白激酶的表达和活化的方法尚未建立。因此，我们开发了一种使用Multi-PK抗体和Phos-tag SDS-PAGE的组合方法来分析细胞内蛋白激酶的表达和磷酸化状态。使用这种新方法，检测了用抑制多种酪氨酸激酶活性的抗癌剂处理的细胞中各种蛋白激酶的表达和磷酸化状态的变化。因此，新方法是一种分析细胞内蛋白激酶的有用技术。
Multi-PK antibody recognizes a wide variety of protein kinases in various species. Using Phos-tag SDS-PAGE, phosphorylated proteins are visualized as slower migration bands compared with corresponding non-phosphorylated proteins. This combined method can be used for detecting changes in the expression and phosphorylation state of various intracellular protein kinases. Protein kinase expression and activity play important roles in diverse cellular functions through regulation of phosphorylation signaling. The most commonly used tools for detecting the protein kinase are protein kinase-specific antibodies, and phosphorylation site-specific antibodies were used for detecting activated protein kinase. Using these antibodies, only one kinase was analyzed at a time, however, a method for analyzing the expression and activation of a panel of protein kinases in cells is not established. Therefore, we developed a combined method using Multi-PK antibody and Phos-tag SDS-PAGE for profiling the expression and phosphorylation state of intracellular protein kinases. Using the new method, changes in the expression and phosphorylation state of various protein kinases were detected in cells treated with anticancer agent which inhibit multiple tyrosine kinase activities. Therefore, the new method is a useful technique for analysis of intracellular protein kinases.