Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE

Protein kinase expression and activity play important roles in diverse cellular functions through regulation of phosphorylation signaling. The most commonly used tools for detecting the protein kinase are protein kinase-specific antibodies, and phosphorylation site-specific antibodies were used for detecting activated protein kinase. Using these antibodies, only one kinase was analyzed at a time, however, a method for analyzing the expression and activation of a panel of protein kinases in cells is not established. Therefore, we developed a combined method using Multi-PK antibody and Phos-tag SDS-PAGE for profiling the expression and phosphorylation state of intracellular protein kinases. Using the new method, changes in the expression and phosphorylation state of various protein kinases were detected in cells treated with anticancer agent which inhibit multiple tyrosine kinase activities. Therefore, the new method is a useful technique for analysis of intracellular protein kinases. • Multi-PK antibody recognizes a wide variety of protein kinases in various species. • Using Phos-tag SDS-PAGE, phosphorylated proteins are visualized as slower migration bands compared with corresponding non-phosphorylated proteins. • This combined method can be used for detecting changes in the expression and phosphorylation state of various intracellular protein kinases.