Isolation of steroid receptor binding protein from chicken oviduct and production of monoclonal antibodies

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Abstract
Previous studies have shown that the molybdate-stablized progesterone receptor from the chick oviduct contains a nonhormone binding component with a MW of 90,000. This protein has also been shown to be associated with some other molybdate-stablized steroid receptors of the oviduct. In order to access this larger pool of the receptor binding protein, an isolation procedure was developed based on the observation that the protein is selectively shed from proteins adsorbed to heparin-agarose when molybdate is removed. The protein obtained by this procedure is shown to be the same as that isolated from affinity-purified progesterone receptor as compared by protease digestion and 1-dimensional peptide mapping. Four IgG secreting hybridoma cell lines were generated against the 90,000-dalton antigen. All of the antibodies recognize the 90,000 dalton protein obtained by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. In addition, 2 of the antibodies complex, the molybdate-stablized progesterone receptor as demonstrated by sedimentation analysis on sucrose gradients. One of these antibodies was used to show the presence of the 90,000-dalton component in molybdate-stablized glucocorticoid and androgen receptors and also to show its presence in brain, liver, and skeletal muscle, but not in serum.