Preparation and characterization of antibodies to o ‐phosphotyrosine and their use for identification of phosphotyrosine‐containing proteins

To facilitate the identification of phosphotyrosine (Ptyr)‐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, 32 P‐labelled A431 cells, and 130kd and 60kd proteins from Rous sarcoma virus (RSV)‐transformed chick cell lysate which had been labelled in vitro , with γ‐ 32 P‐ATP. Immunofluorescent staining of RSV‐transformed cells and A431 cells showed specific localization of P‐tyr‐containing proteins in the cytoplasm, plasma membrane, and nucleoluslike structures. The results demonstrated the usefulness of the antibodies for identification or isolation of P‐tyr‐containing proteins.