Generation and characterization of monoclonal antibodies to the Src-family kinase Hck

Hck, a member of the Src-family of protein tyrosine kinases, is expressed primarily in hematopoietic cells of the myeloid and B-lymphocyte lineages. Hybridoma cell lines were established that secrete monoclonal antibodies (MAbs) to Hck. Three of the MAbs were extensively characterized and designated H7, H34, and H42. The MAbs H7 and H34 recognized an epitope within the SH3 domain of Hck, while the epitope recognized by the H42 MAb resides within the Unique domain. All three MAbs specifically recognized the p59 and p56 isoforms of Hck in transiently transfected 293T cells and in a murine macrophage cell line. Notably, the antibodies did not cross-react with other Src-family kinases tested. Under native conditions, the MAbs H34 and H42 efficiently immunoprecipitated Hck from transfected cells. Both MAbs were also successfully used for the immunofluorescent staining of Hck in intact cells. Thus, the MAbs described herein should be useful in studies of Hck function and expression.