Detection of protein kinase activities toward oligopeptides in sodium dodecyl sulfate-polyacrylamide gel.
In the previous paper, we reported a sensitive method for detection of protein kinase activities in gels after SDS-polyacrylamide gel electrophoresis (Kameshita, I., and Fujisawa, H. (1989) Anal. Biochem. 183, 139-143). This method is useful for the detection of various protein kinase activities toward protein substrates included in gels, but inapplicable to oligopeptide substrates because most of the oligopeptides eluted from the gel matrix during electrophoresis. The present study describes a new procedure for the detection of protein kinase activities toward synthetic oligopeptides in the gel. The oligopeptides which were linked to amino acid polymers such as poly-L-lysine through their amino-terminal cysteinyl residue by a heterobifunctional reagent were efficiently retained in the gel matrix and served as substrates for the protein kinases. As little as 2.5 pg of the catalytic subunit of cAMP-dependent protein kinase was detected by this in-gel assay method using a synthetic peptide as a substrate. This technique can be used for selective and sensitive detection of various protein kinases in crude tissue extracts.
Kameshita
I
I
Department of Biochemistry, Asahikawa Medical College, Asahikawa, 078, Japan.
Fujisawa
H
H
D000180:Acrylic Resins; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity
D000180:Acrylic Resins; D005782:Gels; D009842:Oligopeptides; C016680:polyacrylamide gels; D012967:Sodium Dodecyl Sulfate; D011494:Protein Kinases