Detection of protein kinase substrates in tissue extracts after separation by isoelectric focusing |
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Authors: | Yukako Senga Tadashi Nagamine Mari Sekiguchi Keisuke Kaneko Noriyuki Sueyoshi Isamu Kameshita |
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Institution: | Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan |
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Abstract: | Here we report a simple and useful method to detect endogenous substrates of protein kinases. When crude tissue extracts were resolved by liquid-phase isoelectric focusing (MicroRotofor) and the separated protein fractions were phosphorylated by protein kinases such as Ca2+/calmodulin-dependent protein kinase I or cAMP-dependent protein kinase, various proteins in the different fractions were efficiently phosphorylated. Since a higher number of substrates could significantly be detected using the resolved fractions by MicroRotofor as compared to direct analysis of the original tissue extracts, our present method will be applicable to the screening of endogenous substrates for various protein kinases. |
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