| Assay Method Information | |
| | Alpha-Opioid Receptor GTPgammaS Functional Assay |
| Description: | Functional [35S]GTPgammaS binding assays were conducted as follows. Kappa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/4 kappa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 uL/well) was transferred to 96-shallow well polypropylene plates containing 10 xL of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25 C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Packard) using a 96-well tissue harvester (Packard) and followed by three filtration washes with 200 uL ice-cold binding buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 C. |
| Affinity data for this assay | |
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