Confocal fluorescence microscopy for antibodies against a highly conserved sequence in SH2 domains

We have prepared monoclonal antibodies for a highly conserved sequence (GTFLVRESETTK) in SH2 domains. Mouse IgG1s (12E and 32D) prepared against a peptide-conjugated keyhole lympet hemocyanin specifically bound the antigenic peptide but not the carrier protein. Western blot analysis showed that one IgG1 (12E) recognized mainly 62 kDa proteins (possibly src-family tyrosine kinases) from triton X-100 extracts of RBL-2H3 cells and that another (32D) recognized mainly 32 and 110 kDa proteins. Confocal fluorescence microscopy showed that the SH2 domains had a diffuse cytoplasmic distribution and were not present in the nucleus. Following antigen stimulation, a markedly different cellular distribution was observed in the cells stained with 12E and 32D IgGs. 12E IgGs strongly stained the plasma membranes while 32D IgGs stained small granules in the cytoplasm. As 12E IgGs bound 62 kDa proteins on Western blotting, the results suggest that tyrosine kinases cluster along the plasma membranes and/or than conformational changes occur in the domains after antigen stimulation.