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. 1] Estrogen receptor 21 Cytosol receptor can also be partially purified {ca 4-fold) either before or after covalent labeling by precipitating the receptor with 30% ammonium sulfate. Ammo¬ nium sulfate precipitation also serves to separate the receptor from the bulk of the cytosol proteases, and has proven particularly beneficial with mammary tumor samples. Preparation of cytosol receptors from cells follows a very similar protocol. In order to ensure that receptors in the cells are largely unoccupied, cells (such as MCF- 7 breast cancer cells) which are grown in Eagle's Minimal Essential Medium (MEM) with 5% calf serum are transferred to Eagle's MEM with 5% charcoal dextran- treated calf serum (CDCS) for 7-10 days before harvesting. MCF-7 cell cytosol is prepared by harvesting near-confluent T-150 flasks and washing the cells twice with TEG buffer containing 20 mM sodium molybdate. The cells are then homogenized in the same buffer and the homogenate is centrifuged for 30 min at 180000g. The supernatant (cytosol) is then made 3% in DMF and labeled with 20 nM [^H]Tam-Az (or with 10 nM [^Hjestradiol to determine receptor content of the cytosol) for 2 h at 0-4°C. Determination of non-specific binding is accom¬ plished by including a 200-fold excess of unlabeled estradiol in parallel incubations. After the incubations, excess ligand is removed by incubation with charcoal-dextran slurry (1 part slurry to 9 parts cytosol) for 10 min at 0-4°C, and the charcoal is then removed by centrifugation at 12600g (5 min). The radioactivity associated with receptor after charcoal-dextran treatment at 0°C measures total attachment which may be covalent or non-covalent. Measurement of covalent attachment is deter¬ mined by an ethanol disk assay (see below). C. Labeling of intact cells, subcellular fractionation and preparation of nuclear receptor extracts Near-confluent T-150 flasks of cells in tissue culture media containing 5% CDCS are labeled with 10 nM [^H]E2 or 20 nM [^H]Tam-Az for 1 h at 37°C. If labeling is with pH]KN-Az, 40 nM pH]KN-Az is utilized, and labeling is for 3 h at 37°C. For non¬ specific binding determinations, cells are preincubated for 30 min at 37°C with 200- fold excess of unlabeled E2, although coincubation of [^H]Tam-Az or pH]KN-Az with excess unlabeled E2 gave the same results. Cells are harvested (using Hank's balanced sah solution (HBSS) containing 1 mM EDTA), washed with TEG buffer and then homogenized with 1 ml TEG buffer. The homogenate is centrifuged (800g, 10 min) and the supernatant collected. The crude nuclear pellet is washed twice with TEG buffer (pH7.4) and the supernatants combined. The supernatant fraction is centrifuged at ISOOOOg (30 min), and the resulting supernatant (cytosol) and high speed pellet are assayed for radioactivity. For salt extraction of the nuclear pellet, the washed nuclear pellet from two T-150 flasks of cells is resuspended in 0.5 ml of Tris-extraction buffer (10 mM Tris-HCl, 1.5 mM EDTA, 10% glycerol, 0.6 M NaCl, pH8.5 at 4°C). The suspension is incubated for 1 h at 0-4°C with resuspension every 15 min, and then centrifuged at ISOOOOg for 30 min to give the nuclear salt extract and nuclear pellet fractions. Cells incubated with [^H]Tam-Az in the presence and absence of a 200-fold excess of unlabeled E2 can also be directly lysed with SDS sample buffer (1% SDS, 0.13 M mercaptoethanol, 20% glycerol), thereby avoiding subcellular fractionation.
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