Molybdate stabilizes soluble [H]dexamethasone binding sites in rat brain tissue in incubations at room temperature (22 °C)

We have described an in vitro procedure for the measurement of glucocorticoid receptor concentrations in soluble fractions prepared from rat brain tissue using a relatively short incubation period of 1 h at room temperature (22 °C). Using [³H]dexamethasone as ligand we found that, with the addition of sodium molybdate (Na₂MoO₄) to the buffer, estimates of receptor-binding parameters derived from Scatchard analysis (K_dandB_max) did not differ significantly between assays using 4–5 h incubations at 0–4 °C and those using a 1 h incubation performed at room temperature (22 °C). The use of molybdate was critical; 1 h incubations at 22 °C in the absence of molybdate resulted in a substantial ( 65%) loss of [³H]dexamethasone binding.