The purified ERα LBDs of 5 nM were incubated with 10 nM [3H]-E2 with varying concentrations (0.1 nM to 10 μM) of competitors (LAS and 4-hydroxytamoxifen [4-OHT]) in binding buffer (10 mM Tris pH 7.6, 300 mM NaCl, 5 mM EDTA, 10% glycerol, 1 mM DTT). After a 1-h incubation on ice, 50 μL of each reaction was loaded onto a CPG column at 4 °C in triplicate and incubated for 5 min. The columns were then washed with approximately 10 mL wash buffer (10 mM Tris pH 7.4, 400 mM NaCl) to remove unbound ligands. Remaining [3H]-E2 was eluted with 1 mL ethanol and counted in a liquid scintillation counter. Data were analyzed as described previously [13, 31].
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