4.4. [35S]GTPγS Binding Assays for Opioid Receptors

Binding of [³⁵S]GTPγS to membranes from CHO stably expressing the human opioid receptors was conducted according to the published procedure [26]. Cell membranes (5–10 µg) in Buffer A (20 mM HEPES, 10 mM MgCl₂, and 100 mM NaCl, pH 7.4) were incubated with 0.05 nM [³⁵S]GTPγS, 10 µM GDP and various concentrations of test compounds in a final volume of 1 mL for 60 min at 25 °C. Non-specific binding was determined using 10 µM GTPγS, and the basal binding was determined in the absence of test ligand. Samples were filtered over Whatman GF/B glass fiber filters and counted as described for competitive binding assays. All experiments were performed in duplicate and repeated three times with independently prepared samples.