Phosphate-binding tag, a new tool to visualize phosphorylated proteins
Phosphate-binding tag, a new tool to visualize phosphorylated proteins
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DOI：
10.1074/mcp.t500024-mcp200
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发表时间：
2006-04-01
影响因子：
7
通讯作者：
Koike, T
中科院分区：
文献类型：
--
作者：
Kinoshita, E;Kinoshita-Kikuta, E;Koike, T
我们介绍了两种方法的可视化磷酸化的蛋白质，使用烷氧基桥双核金属（即Zn 2+或Mn 2+）配合物作为新的磷酸结合标签（Phos标签）分子。Zn 2 +-和Mn 2 +-Phos-标记分子都优先捕获与Ser、Thr和Tyr残基结合的磷酸单酯二价阴离子。一种方法是基于ECL系统，使用生物素悬垂的Zn 2 +-Phos-标签和辣根过氧化物酶缀合的链霉亲和素。我们证明了蛋白质磷酸化状态的磷酸选择性ECL信号的电印迹分析。另一种方法是基于磷酸化蛋白质在聚丙烯酰胺结合的Mn 2 +-Phos-标签的SDS-PAGE中的迁移率变化。与相应的去磷酸化蛋白质相比，凝胶中的磷酸化蛋白质被可视化为较慢的迁移带。我们通过磷酸盐亲和电泳（Mn 2 +-Phos-tag SDS-PAGE）证明了激酶和磷酸酶测定。
We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn2+ or Mn2+) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn2+- and Mn2+-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn2+-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn2+-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn2+-Phos-tag SDS-PAGE).