Detection of protein kinase substrates in tissue extracts after separation by isoelectric focusing.
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 Ca(2+)/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.
Senga
Yukako
Y
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
Nagamine
Tadashi
T
Sekiguchi
Mari
M
Kaneko
Keisuke
K
Sueyoshi
Noriyuki
N
Kameshita
Isamu
I
D000818:Animals; D001921:Brain / Q000201:enzymology; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1 / Q000737:chemistry* / Q000302:isolation & purification; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification; D007525:Isoelectric Focusing / Q000379:methods*; D010766:Phosphorylation; D051381:Rats; D013379:Substrate Specificity; D014020:Tissue Extracts
D014020:Tissue Extracts; D017868:Cyclic AMP-Dependent Protein Kinases; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1