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Sugiyama Y, Katayama S, Kameshita I, et al. Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE. MethodsX. 2015;2:469-74doi: 10.1016/j.mex.2015.11.007.
Sugiyama, Y., Katayama, S., Kameshita, I., Morisawa, K., Higuchi, T., Todaka, H., Kinoshita, E., Kinoshita-Kikuta, E., Koike, T., Taniguchi, T., & Sakamoto, S. (2015). Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE. MethodsX, 2469-74. https://doi.org/10.1016/j.mex.2015.11.007
Sugiyama, Yasunori, et al. "Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE." MethodsX vol. 2 (2015): 469-74. doi: https://doi.org/10.1016/j.mex.2015.11.007
Sugiyama Y, Katayama S, Kameshita I, Morisawa K, Higuchi T, Todaka H, Kinoshita E, Kinoshita-Kikuta E, Koike T, Taniguchi T, Sakamoto S. Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE. MethodsX. 2015 Nov 19;2:469-74. doi: 10.1016/j.mex.2015.11.007. eCollection 2015. PMID: 26844212; PMCID: PMC4703585.
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MethodsX. 2015 Nov 19;2:469-74. doi: 10.1016/j.mex.2015.11.007. eCollection 2015.

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

MethodsX

Yasunori Sugiyama, Syouichi Katayama, Isamu Kameshita, Keiko Morisawa, Takuma Higuchi, Hiroshi Todaka, Eiji Kinoshita, Emiko Kinoshita-Kikuta, Tohru Koike, Taketoshi Taniguchi, Shuji Sakamoto

Affiliations

  1. Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
  2. Laboratory of Molecular Biology, Science Research Center, Kochi Medical School, Kochi 783-8505, Japan.
  3. Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.

PMID: 26844212 PMCID: PMC4703585 DOI: 10.1016/j.mex.2015.11.007

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.

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

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