Affinity labelling and cloning of steroid and thyroid hormone receptors / edited by H. Gronemeyer.

Date:
[1988]
Catalogue details

Licence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Credit: Affinity labelling and cloning of steroid and thyroid hormone receptors / edited by H. Gronemeyer. Source: Wellcome Collection.

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    Ch. 2] Glucocorticoid receptor 41 ammonium sulfate precut was used to decrease the amount of non-receptor proteins present. After 30 min of incubation at 0°C, the precipitation was collected by centrifugation at 2320g for 10 min at 0°C. The precipitate was resuspended in a minimal volume of pH 8.8 TAPSq buffer containing 20 mM sodium molybdate to preserve the unactivated status of the complexes (Reichman et al., 1984) and stored at -20°C. Recovery of covalent receptor-Dex-Mes complexes after ammonium sulfate precipitation was generally greater than 95%. It has been reported that the efficiency of 'whole cell' covalent labeling of HeLa S3 cell receptors at 0°C by [^H]Dex-Mes is increased from 10% to greater than 90% if the pH of the homogenization buffer is raised from 8.0 to 9.0 (Cidlowski and Richnon, 1984). Assuming that the higher pH does not destroy the steroid binding of the HeLa S3 receptors (с/. Section III.C, and Simons et al., 1983), this indicates that most of the covalent labehng of the whole cell receptor occurs after cell rupture. In contrast, Miller and Simons (in preparation) find no difference (±7%) in the absolute amount of Fu5-5 cell receptors that are labeled in whole cells at 0°C when the pH of the lysis buffer (see above) is varied from 6.8 to 9.5. IX. PARTIAL PURIFICATION OF UNACTIVATED AFFINITY LABELED RECEPTORS A. (N114)2804 precipitation of [^H]Dex-Mes labeled receptors (Reichman et al.^ 1984) HTC cell cytosol receptors in TAPS buffer (final pH, ~8.8 at 0°C; see Section III.C) containing 20 mM Na2Mo04 were labeled with pH]Dex-Mes ± 80-fold excess of [^HJdexamethesone for 2.5 h at 0°C. The solutions were then adjusted to contain 50 mM ß-mercaptoethanol (to consume unreacted Dex-Mes (Simons et al., 1983)) and 80-fold excess of [^H]dexamethesone in both the competed and the uncompeted samples (to prevent possible binding of [^H]Dex-Mes reaction products). After a further 45 min incubation at 0°C, 0.65 volumes of a saturated (N114)2804 solution (in water; pH adjusted to —8.6 at 0°C with concentrated NH4OH) was rapidly added (final (N114)2804 concentration, 39.4%). After incubation at 0°C for 30 min, the precipitate was centrifuged (0°C for 1 min in a microfuge), resuspended in pH 8.2 TAPSo buffer (25 mM TAPS, 1 mM EDTA, 10% (v/v) glycerol, pH adjusted at 0°C) containing 20 mM Na2Mo04, quick frozen at -78°C, and stored at -20°C or -78°C. By the criterion of binding to DNA-cellulose columns in pH 8.2 TAPSq buffer containing 20 mM Na2Mo04, (N114)2804 precipitated, unactivated HTC cell com¬ plexes contain <2% activated complexes. The recovery of precipitated, pH]Dex-Mes labeled receptor is very high (>90% ; S.S. Simons, Jr., et al., unpublished results). Chromatography of the labeled receptors on Sephadex G-lOO or G-150 prior to (NH4)2S04 precipitation reduces the final recovery of labeled receptors to ~65% with no appreciable further purification (8. 8. Simons, Jr., preliminary results). B. Immunoadsorption of [^H]Dex-Mes labeled receptors (Eisen et al., 1981) Polyclonal rabbit anti-rat glucocorticoid receptor antibody (IgG) (Eisen, 1980) was purified from sera by chromatography on protein A-Sepharose. The purified IgG
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