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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    38 Glucocorticoid receptor [Ch. 2 some of the non-specifically labeled proteins are more extensively labeled in the presence of competing [^HJdexamethasone (Fig. 3 and Simons et al., 1983). To date, SDS-polyacrylamide gel electrophoresis has proved to be the only method that can reliably be used to quantitate covalently labeled receptors. However, even this method does not work with very high levels of [^H]Dex-Mes (e.g. 2= 10^ M in whole HTC cell labeling experiments) because the non-specific labeling in the region of the 98K receptor is so extensive (S. S. Simons, Jr., eia/., unpublished results). A. Preparation of samples (Simons et а/. Д983) For samples containing large amounts of pH]Dex-Mes labeled receptors (i.e. ^5000 dpm/20 fjLÌ), the sample is diluted with an equal volume of 2xSDS sample buffer (0.6 M Tris (pH 8.85), 2% SDS, 0.2 M dithiothreitol, 20% (v/v) glycerol, and bromphe- nol blue). Dilute solutions of labeled receptor are concentrated by precipitation in 10% trichloroacetic acid (TCA) at 0°C for at least 2 h (samples can be left overnight at 0°C at this stage). If the total protein concentration after addition of TCA is <100 /Ltg/ml, soybean trypsin inhibitor (at a final concentration of 100 /i-g/ml) is added as carrier protein. The precipitate is collected by centrifugation (2000g for 10 min), resuspended in 1 ml of 10% TCA for at least 1 h, and recentrifuged as before, all at 0°C. The supernatant is removed by aspiration to give pellets which can be stored at 0 to — 20°C for a few days. To prepare the acid-precipitated material for Polyacryla¬ mide gels, the pellet is dissolved in a minimum amount of 1N NaOH (usually 10-15 /Ltl) and then diluted with up to 100 of 2xSDS sample buffer. Acetone has sometimes been used to wash acid-precipitated pellets (Wrange et ai, 1979). However, we have found that TCA precipitated, covalently labeled receptors are quite soluble in acetone, even at 0°C. Thus, for directly applied samples, the recovery of specific [^H]Dex-Mes labeled receptor at Мг=98 ООО on denaturing Polyacrylamide gels was 5— to 6-fold higher than for acid-precipitated- acetone-washed samples of the same incubation solution (e.g. 6000 dpm vs. 900 dpm). Cytosol solutions can be stored at -20°C, or -78°C, for several months before gel analysis. However, once 2xSDS sample buffer is added to the samples, the abundant proteins and labeled receptor undergo considerable degradation over time, even when stored at — 20°C (S.S. Simons, Jr., unpublished results). B. SDS-polyacrylamide gel electrophoresis of [^H]Dex-Mes labeled receptors (Reichman etal., 1984; Simons, 1987) Samples were diluted with an equal volume of 2xSDS sample buffer, heated at 100°C for 5 min, and appHed to constant percentage acrylamide gels (10.5-11% or 15% overlaid with a 3.6% stacking gel, both with a 1:38.9 ratio of bis-acrylamide to acrylamide), which were run in a water-cooled (15°C) Protean or Protean II slab gel apparatus (Bio-Rad). The gels were run at constant current (20 m A/gel while in the stacking gel, then 30 mA/gel for the 10.5-11% gels and 25 mA/gel for the 15% gels). Gels were fixed and stained in 50% methanol-7.5% acetic acid containing 0.02% Coomassie Blue (R-250), destained in 10% methanol-7.5% acetic acid, incubated for 1 h in Enhance (New England Nuclear) and 30-60 min in 10% Carbowax PEG 8000 (formerly PEG 6000; Fisher; recommended by New England Nuclear) with constant shaking at room temperature, dried on a Bio-Rad model 443 slab gel drier at
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