Development and characterization of polyclonal antibodies against a conserved sequence in the catalytic domain of protein kinases.
Using a synthetic oligopeptide (CGGGTPEYLAPEGGK) crosslinked to keyhole limpet hemocyanin we have raised polyclonal rabbit antibodies against a 9 residue homologous region found in the catalytic domain of most protein kinases. These antibodies reacted during Western immunoblotting with cAMP dependent protein kinase catalytic subunit, phosphorylase kinase gamma subunit and calcium calmodulin dependent protein kinase II which have homologous sequences of GTPEYLAPE, GTPSYLAPE and GTPGYLSPE, respectively. Five other protein kinases did not react with anti-GTPEYLAPE antibodies during Western immunoblotting. Affinity-purified antibodies were able to detect as little as 50 ng of cAMP dependent protein kinase and 200 ng of Ca2+/calmodulin dependent protein kinase II. Immunoblotting of A431 cell plasma membrane vesicles indicated the presence of an approximately 55 kDa protein that contains the conserved sequence and is likely to be a protein kinase. Antibodies directed against conserved sequences present in protein kinases, or possibly other enzymes, may be useful in identifying previously uncharacterized enzymes at the protein level.
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232.
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Hagedorn
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Tettelbach
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D000595:Amino Acid Sequence; D000906:Antibodies / Q000302:isolation & purification; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D000939:Epitopes / Q000032:analysis; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis*; D011494:Protein Kinases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D000906:Antibodies; D000939:Epitopes; D046911:Macromolecular Substances; D009842:Oligopeptides; D011494:Protein Kinases