Preparation and characterization of antibodies to O-phosphotyrosine and their use for identification of phosphotyrosine-containing proteins.

To facilitate the identification of phosphotyrosine (P-tyr)-containing proteins, rabbit polyclonal antibodies and mouse monoclonal antibody specifically reactive to P-tyr were prepared by hyperimmunizing the animals with P-tyr-conjugated bovine serum albumin or poly-L-lysine. As determined by a solid-phase radioimmunoassay and an enzyme-linked immunosorbent assay, the antibodies reacted with P-tyr-conjugated target antigens but not with those conjugated with phosphoserine (P-ser) or phosphothreonine (P-thr). This immune reaction was strongly blocked by 2 mM P-tyr and phenylphosphate but not by P-ser or P-thr. The antibodies were capable of isolating, as the major P-tyr-containing components, a 170kd protein (most likely the EGF receptor) from EGF-stimulated, 32P-labelled A431 cells, and 130kd and 60kd proteins from Rous sarcoma virus (RSV)-transformed chick cell lysate which had been labelled in vitro with gamma-32P-ATP. Immunofluorescent staining of RSV-transformed cells and A431 cells showed specific localization of P-tyr-containing proteins in the cytoplasm, plasma membrane, and nucleolus-like structures. The results demonstrated the usefulness of the antibodies for identification or isolation of P-tyr-containing proteins.