4.3 Article

Kinase activity-tagged western blotting assay

期刊

BIOTECHNIQUES
卷 68, 期 4, 页码 211-213

出版社

FUTURE SCI LTD
DOI: 10.2144/btn-2019-0136

关键词

cell signaling; laboratory safety; nonradioactive assay; phosphorylation; post-transcriptional modification; protein kinases; western blotting

资金

  1. JSPS KAKENHI [JP19K06573, JP19H03125]
  2. Society for Research on Umami Taste
  3. Ehime prefecture

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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 SUMMARY Kinase 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.

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