Use of a monoclonal antibody (APE-1) directed against the conserved GTPEYLAPE motif present in protein kinases as an affinity purification reagent.
cDNA sequence studies have predicted the existence of protein kinases that have not been characterized at the protein level. This laboratory has previously shown that polyclonal rabbit antibodies directed against a highly conserved sequence motif, GTPEYLAPE, present in the catalytic domain of many protein kinases will react during immunoblots with several protein kinases containing the same or homologous motifs. In this report I describe the development of a mouse monoclonal antibody, designated APE-1, that reacts with the GTPEYLAPE motif. In addition, the use of this monoclonal antibody in the affinity purification of kinase activities from rabbit reticulocyte lysate is described. This approach may provide a general method for the purification of previously uncharacterized protein kinases that share regions which are homologous to the GTPEYLAPE motif. Refinements in this method may permit the rapid purification of nonabundant and/or rapidly inactivated protein kinases that have not been isolated using other approaches.
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232.
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Hagedorn
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D051379:Mice; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D011494:Protein Kinases