Molybdate stabilizes soluble [3H]dexamethasone binding sites in rat brain tissue in incubations at room temperature...

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 degrees 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 (Kd and Bmax) did not differ significantly between assays using 4-5 h incubations at 0-4 degrees C and those using a 1 h incubation performed at room temperature (22 degrees C). The use of molybdate was critical; 1 h incubations at 22 degrees C in the absence of molybdate resulted in a substantial (approximately 65%) loss of [3H]dexamethasone binding.