Supporting information for Zhu et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0336132100

Initially, various detergents and procedures were evaluated for their ability to solubilize the receptor from fish ovaries while retaining specific progestin binding activity. Treatment with 12 mM Triton X-100 for 30 min followed by removal of the detergent with polyabsorbents for 2 hr resulted in efficient extraction of the receptor (79%) from the membrane fraction and the appearance of significant amounts of high affinity (Kd 8.7 nM), saturable (B_max = 0.204 pmol·g^–1 ovary) and displaceable specific 20β-S binding in the solubilized fraction (data not shown).

DEAE chromatography with a stepwise NaCl gradient resolved the solubilized ovarian proteins into three UV-absorbing peaks (Fig. 8a). Fractions in each protein peak were pooled and further purified and concentrated to 2 mg of protein per ml with a 10,000-Da molecular weight cutoff centrifuge concentrator. Other column fractions did not bind progestins above background levels and therefore were discarded. Proteins with molecular weights >10 kDa were collected, and their progestin binding activities were analyzed. Although specific progestin binding was detected in all three fractions, approximately half of the total specific binding was concentrated in fraction 1 (Fig. 8a). Analyses of the partially purified proteins by SDS/PAGE and silver staining showed only minor amounts of protein in fraction 1 with a major band at 40 kDa, whereas the two other fractions contained larger quantities of protein with multiple bands present on the SDS gel (Fig. 8b). These results suggested the minor protein in fraction 1 was enriched with the progestin membrane receptor. Therefore, this partially purified receptor fraction was used to immunize mice.

Seventeen partially cloned positive antibodies were first selected based on the presence of IgGs and positive immunoreactions with solubilized ovarian membrane proteins by screening with ELISA. Eight of these antibodies were further selected based on positive immunoreactive bands in the 25- to 100-kDa range identified by Western blot analysis. Thereafter, these antibodies were further cloned and screened with a double-antibody receptor capture assay (1). Three positive monoclonal antibodies were finally identified based on specific binding to the progestin (Table 1). A band ≈40 kDa was detected in the solubilized membrane protein fraction using monoclonal antibody PR10-1 (Fig. 8c). No antibody binding was detected in the cytosolic fraction. A single major band ≈40 kDa was also detected in purified membrane fraction 1 (Fig. 8c).

1. Thomas, P., Zhu, Y. & Pace, M. (2002) Steroids 67, 511–517.