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.

    26/332 page 22
    22 Estrogen receptor [Ch. 1 Cells are incubated for 10 min or 30 min at room temperature with SDS sample buffer, and these whole cell extracts are then boiled for 2 min at 100°C and analyzed by SDS-polyacrylamide gel electrophoresis. The estrogen receptors in intact MCF-7 cells can also be effectively labeled with [^H]Tam-Az not only when cells are attached to the flasks but also in cells in suspension. Since less volume and, hence, less radiolabeled Tan-Az is needed for suspension labehng, this protocol is used frequently. For labeling of cells in suspension, MCF-7 cells are released from the flask with HBSS containing 1 mM EDTA, decanted into a 50 ml conical tube, and pelleted at 1000 rpm for 8 min. The intact cells are resuspended in MEM+5% CDCS growth media and labeled in suspension with 10 nM [^HJestradiol or 20 nM [^H]Tam-Az with and without a 200- fold excess of unlabeled estradiol for 1 h at 37°C. The cells are then washed with TEG buffer and homogenized in 10 mM Tris, 1.5 mM EDTA, 10% glycerol buffer, pH 7.4 (TEG buffer), with a Dounce homogenizer using a B-pestle. The homogenate is centrifuged at 800^ for 10 min, washed once in TEG buffer, and the resulting nuclear pellet is extracted for 1 h at 0-4°C with TEG buffer (pH 8.5) containing 0.6 M NaCl, with resuspension every 15 min. The suspension is then centrifuged at 180000g for 30 min to give the nuclear salt-extracted receptor preparation. D. Assay of covalent attachment Attachment is assayed by an ethanol disk assay (Katzenellenbogen et al., 1977b). For the ethanol disk assay, samples of 50 /al or less are spotted onto 2.2 cm Whatman 3 filter paper disks. These are collected and washed in boiling 95% ethanol twice for 15 min, rinsed twice at room temperature in 1:1 ether:95% ethanol, rinsed twice in pure ether and dried; all wash and rinse solutions are used at 10 ml/disk. The protein, together with any covalently attached compound, is precipitated by the ethanol and is retained within the fibres of the paper, while the non-covalently attached material is extracted from the disk. The disks are counted in mini-vials in 5 ml aqueous counting solution (ACS) scintillation cocktail (Amersham). E. Scintillation counting All samples in which [^H]Tam-Az or [^H]KN-Az are protein bound are counted in ACS scintillation cocktail (Amersham). This cocktail proved superior in solubilizing proteins which otherwise remained in an aqueous phase and were counted only at low efficiency. [^HJestradiol-labeled samples are counted in triton-xylene scintilla¬ tion cocktail (Katzenellenbogen et al., 1973a). When counting was carried out in our Nuclear-Chicago Isocap 3000 refrigerated scintillation counter, an efficiency of 33-45% was obtained. III. ANALYSIS OF COVALENTLY LABELED ESTROGEN RECEPTORS A. Analysis of receptor size by SDS-polyacrylamide gel electrophoresis Samples to be analyzed, after mixing with an equal volume of 2-fold concentrated SDS-sample buffer, are incubated at 100°C for 2 min. The 2-fold concentrated sample buffer for electrophoresis contains 0.125 M Tris-HCl (pH6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol and bromophenol blue. Gels either are prepared according to Laemmli (1970) and contain 10% total acrylamides (T) with
    digitised image 1
    digitised image 2
    digitised image 3