Radioligand Binding Assay Protocol

Radioligand binding was performed as previously described [40 (link),41 (link)]. For the binding assay 50 µL of a dilution series of peptide was added to 50 µL of 3.3 nM [³H]DPDPE (K_d = 3.87 nM) or 2.35 nM of [³H]DAMGO (K_d = 1.07 nM) or 0.8 nM of [³H]U69,593 (K_d = 1.2 nM) in a clear 96 well plate. Next, 100 µL of membrane suspension containing 7 µg protein was added to the agonist wells and incubated for 90 min at room temperature. The reaction mixture was then filtered over a GF-B filter plate (Perkin Elmer) followed by four quick washes with ice-cold 50 mM Tris HCl. The plate was dried overnight, after which 50 µL scintillation fluid (Ultimagold uLLT) was added and radioactivity was counted on a Packard TopCount NXT scintillation counter. All working solutions were prepared in a radioligand assay buffer containing 50 mM Tris HCl, 10 mM MgCl₂, and 1 mM ethylenediaminetetraacetic acid at pH 7.4.