Assay for Opioid Receptor Binding

Radioligands [³H]U69,593 (49.3 Ci/mmol), [³H]diprenorphine (33.9 Ci/mmol) and [³⁵S]GTPγS (1250 Ci/mmol) were purchased from PerkinElmer (Boston, MA, USA). Guanosine diphosphate (GDP), GTPγS, U69,593, U50,488, diprenorphine, tris(hydroxymethyl) aminomethane (Tris), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), bovine serum albumin (BSA), formalin, nor-BNI and cell culture media and supplements were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). All other chemicals were of analytical grade and obtained from standard commercial sources. Compound A was obtained from Maybridge Chemical Co., Ltd. (Cornwall, UK) as in [50 (link)], and was prepared as 1 mM stock in 0.5% acetic acid solution and further diluted to working concentrations in the appropriate medium.