Structural analysis of chicken oviduct progesterone receptor using monoclonal antibodies to the subunit B protein.
Two monoclonal antibodies against the B subunit (Mr 108 000) of chick oviduct progesterone receptor (PgR) were produced by immunizing rats and fusing spleen cells with NS-1 mouse myeloma cells. The hybridoma lines designated 9G10 and 3E8 produce rat IgG2a and IgG2b, respectively. Antibody-receptor interactions were demonstrated under protein denaturing conditions. Previous studies by Weigel et al. [Weigel, N. L., Tash, J. S., Means, A. R., Schrader, W. T., & O'Malley, B. W. (1981) Biochem. Biophys. Res. Commun. 102, 513-519] have shown that chick PgR can be phosphorylated in vitro. Both antibodies, 9G10 and 3E8, were shown to displace partially denatured 32P-labeled PgR from its characteristic 4S position on high salt sucrose density gradients to a form with a higher sedimentation coefficient. Further specificity and sensitivity were demonstrated by protein immunoblotting experiments. In partially purified as well as electrophoretically pure receptor B subunit preparations antibodies reacted with the Mr 108 000 receptor B band. By immunoblot assay 9G10 was 20-fold more sensitive than 3E8, the former detecting down to 5 ng of receptor and the latter 100 ng. Because of its sensitivity 9G10 was able to detect the Mr 108 000 receptor as a single band in a crude oviduct fraction and did not cross-react with any other contaminating proteins. Receptor antigenic determinants were localized by immunoblot assay of receptor proteolytic digests.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal; D002499:Centrifugation, Density Gradient; D000939:Epitopes / Q000032:analysis; D005260:Female; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010057:Oviducts / Q000032:analysis*; D011487:Protein Conformation; D011980:Receptors, Progesterone / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D011980:Receptors, Progesterone