title: Purification of denatured epidermal growth factor-receptor from A431 human epidermoid carcinoma cells.
abstract: A rapid and simple method was developed for isolating denatured epidermal growth factor (EGF)-receptor suitable for use in preparation of polyclonal antisera. Membranes from A431 cells (which possess unusually high numbers of EGF-receptors) were phosphorylated in vitro with [gamma-32P]ATP and run on preparative sodium dodecyl sulfate (SDS)-polyacrylamide gels. The Mr 170,000 major phosphorylated region was excised from the gels, eluted, and protein chromatographed on SDS-hydroxylapatite. Fractions containing the Mr 170,000 tyrosine-phosphorylated protein were pooled, concentrated, and rerun on preparative SDS gels. The protein eluted from these gels was judged to be highly purified, based on peptide mapping and on comparison of proteins immunoprecipitated by monoclonal antibody against the EGF-receptor with proteins precipitated by polyclonal antibody prepared against the Mr 170,000 protein described here. The polyclonal antiserum recognized native and denatured EGF-receptor from human, rat, and mouse cells and should prove useful in studying EGF-receptor synthesis and function.
mesh_terms: D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000032:analysis; D002460:Cell Line; D066246:ErbB Receptors; D006801:Humans; D007106:Immune Sera; D051379:Mice; D009363:Neoplasm Proteins / Q000302:isolation & purification; D011489:Protein Denaturation; D011817:Rabbits; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification*
chemical_list: D007106:Immune Sera; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; D066246:ErbB Receptors