UNIT 18.12 Detection of MAP Kinase Signaling

Elucidation of intracellular signaling pathway is essential to understanding the regulatory mechanisms of cellular function. Mitogen-activated protein kinase (MAPK) signaling cascades are a major pathway in intracellular signaling. The methods used to detect cascade activation involve measurements of protein kinase activities. This unit describes an assay for specific immunoprecipitation of a protein kinase and detection of phosphorylation of the appropriate substrate, an in-gel kinase assay for kinases of unknown identity or for which there are no specific antibodies, and the JNK assay, which involves a broad-based immunoaffinity purification followed by phosphorylation.