4.2.6. GTPγS Binding Assay

The method for measuring agonist-stimulated [³⁵S]-GTPγS binding to the human CB₁ and CB₂ receptors was used as described previously.²⁴ In brief, binding reactions were carried out in 96-well microplates in a final volume of 500 μL. Cell membranes (20 μg) were incubated with 0.5 nM [³⁵S]-GTPγS, 30 μM GDP, and compounds in assay buffer (50 mM Tris-HCl, 150 mM NaCl, 9 mM MgCl₂, 0.2 mM EGTA, and 1.4 mg/mL BSA, pH 7.4) for 2 h at 37 °C with gentle shaking. The nonspecific binding (NSB) was determined using 40 mM nonradiolabeled guanosine 5′-(γ-thio) triphosphate (GTPγS) (PerkinElmer, Waltham, MA). The positive control was attained by utilizing 10 μM unlabeled CP55,940 for the test compound. The reaction was terminated by rapid vacuum filtration, and the membranes were harvested onto a PerkinElmer Unifilter GF/B-96 filter plate and washed three times with ice-cold washing buffer (10 mM Tris-HCl, pH 7.4), and the bound radioactivity was quantified by a Packard TopCount Scintillation Counter.