Kinase activity-tagged western blotting assay
Kinase activity-tagged western blotting assay
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DOI：
10.2144/btn-2019-0136
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发表时间：
2020-04-01
期刊：
影响因子：
2.7
通讯作者：
Takeya, Kosuke
中科院分区：
文献类型：
--
作者：
Eto, Masumi;Katsuki, Shuichi;Takeya, Kosuke
确定蛋白激酶的细胞活性是表征病理生理细胞信号通路的基本步骤。在这里，我们优化了一种非放射性方法，该方法通过SDS-PAGE分离并转移到聚偏氟乙烯膜上后检测组织或细胞中的蛋白激酶。该方法，激酶活性标记的蛋白质印迹法（KAT-WB），由五个步骤组成：电泳的细胞提取物，含有蛋白激酶，电印迹蛋白到聚偏氟乙烯膜，变性-复性，磷酸化，有或没有添加底物蛋白和免疫检测使用抗磷酸特异性抗体。KAT-WB检测到一个Tyr-激酶的自磷酸化和多个激酶对添加的底物的位点特异性磷酸化。KAT-WB测定使我们能够询问多个激酶信号转导通路，而不使用放射性ATP.METHOD SUMMARYKinase活性标记的蛋白质印迹法的目的是分离和检测多个激酶磷酸化本身或特定的底物。印迹上的激酶用胍变性，然后逐步复性，然后用非放射性ATP磷酸化并用磷酸特异性抗体检测。
Determining cellular activities of protein kinases is a fundamental step for characterizing pathophysiological cell signaling pathways. Here, we optimized a non-radioactive method that detects protein kinases in tissues or cells after separation by SDS-PAGE and transfer onto polyvinylidene fluoride membranes. The method, kinase activity-tagged western blotting (KAT-WB), consists of five steps: electrophoresis of cell extracts that contain protein kinases, electroblotting proteins onto polyvinylidene fluoride membrane, denaturation-renaturation, phosphorylation, with or without an added substrate protein and immunodetection using anti-phospho-specific antibodies. KAT-WB detected autophosphorylation of one Tyr-kinase and site-specific phosphorylation of added substrate by multiple kinases. KAT-WB assay enables us to interrogate multiple kinase signaling pathways without using radioactive ATP.METHOD SUMMARYKinase activity-tagged western blotting is designed to separate and detect multiple kinases that phosphorylate themselves or a specific substrate. Kinases on the blot are subjected to denaturation with guanidine and then stepwise renaturation, prior to phosphorylation using nonradioactive ATP and detection with phosphospecific antibodies.