Department of Functional Molecular Science, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Methods Mol Biol. 2021;2261:73-78. doi: 10.1007/978-1-0716-1186-9_6.
We describe a standard protocol for phosphate-affinity fluorescent gel staining that uses a fluorophore-labeled dizinc(II) complex of a derivative of the phosphate-binding tag molecule Phos-tag to detect His- and Asp-phosphorylated proteins separated by SDS-PAGE. The procedure permits the quantitative monitoring of phosphorylated histidine kinases (His-phosphoproteins) and their cognate phosphorylated response regulators (Asp-phosphoproteins) in bacterial two-component signaling transduction systems. The total time required for each gel staining operation is about 2 h at room temperature.
我们描述了一种使用荧光标记的二锌（II）配合物的磷酸盐亲和荧光凝胶染色的标准方案，该配合物是磷酸盐结合标签分子 Phos-tag 的衍生物，用于检测 SDS-PAGE 分离的 His-和 Asp-磷酸化蛋白质。该程序允许在细菌双组分信号转导系统中定量监测磷酸化组氨酸激酶（His-磷酸蛋白）及其同源磷酸化响应调节剂（Asp-磷酸蛋白）。每个凝胶染色操作所需的总时间约为 2 小时，在室温下进行。
