Development and characterization of polyclonal antibodies against a conserved sequence in the catalytic domain of protein kinases

[摘要]

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 γ subunit and calcium calmodulin dependent protein kinase II which have homologous sequences of GTPEYLAPE, GTPYLAPE and GTPYLPE, 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.

[学科分类] 生物化学/生物物理

[关键词] cAMP dependent protein kinase;Phosphorylase kinase;Ca2+/calmodulin kinase II;A431 cell;EDC;l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;sulfo-NHS;N-hydroxysuccinimide;KLH;keyhole limpet hemocyanin;PBS;Dulbecco's phosphate-buffered saline;NBT;4-nitro blue tetrazolium;BCIP;5-bromo-4-chloro-3-indolyl phosphate