title: Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE.
abstract: 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.
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors: Yasunori
authors: Y
authors: Sugiyama
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors: Syouichi
authors: S
authors: Katayama
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors: Isamu
authors: I
authors: Kameshita
authors: Laboratory of Molecular Biology, Science Research Center, Kochi Medical School, Kochi 783-8505, Japan.
authors: Keiko
authors: K
authors: Morisawa
authors: Laboratory of Molecular Biology, Science Research Center, Kochi Medical School, Kochi 783-8505, Japan.
authors: Takuma
authors: T
authors: Higuchi
authors: Laboratory of Molecular Biology, Science Research Center, Kochi Medical School, Kochi 783-8505, Japan.
authors: Hiroshi
authors: H
authors: Todaka
authors: Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
authors: Eiji
authors: E
authors: Kinoshita
authors: Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
authors: Emiko
authors: E
authors: Kinoshita-Kikuta
authors: Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
authors: Tohru
authors: T
authors: Koike
authors: Laboratory of Molecular Biology, Science Research Center, Kochi Medical School, Kochi 783-8505, Japan.
authors: Taketoshi
authors: T
authors: Taniguchi
authors: Laboratory of Molecular Biology, Science Research Center, Kochi Medical School, Kochi 783-8505, Japan.
authors: Shuji
authors: S
authors: Sakamoto
keywords: Erk, extracellular-signal-regulated protein kinase; Kinome; Multi-PK antibody; PKA, cAMP-dependent protein kinase; Phos-tag; Phosphorylation Signaling; Protein kinase; λPPase, lambda protein phosphatase