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.
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001358:Avian Sarcoma Viruses; D002471:Cell Transformation, Neoplastic; D002642:Chick Embryo; D066246:ErbB Receptors; D005260:Female; D005455:Fluorescent Antibody Technique; D051379:Mice; D010761:Phosphorus Radioisotopes; D019000:Phosphotyrosine; D011506:Proteins / Q000032:analysis*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000032:analysis; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology
D000906:Antibodies; D010761:Phosphorus Radioisotopes; D011506:Proteins; D011956:Receptors, Cell Surface; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors