| Assay Method Information | |
| | Mu-Opioid GTPgammaS Binding Assay |
| Description: | [35S]GTP gamma S functional assays were conducted using freshly thawed mu-receptor membranes. Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTP gamma S (0.20 nM; NEN). The prepared membrane solution (1904/well) was transferred to 96-shallow well polypropylene plates containing 10 uL of 20x concentrated stock solutions of the agonist DAMGO ([D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin) prepared in DMSO. Plates were incubated for 30 min at about 25 C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Packard, Meriden, Conn.) using a 96-well tissue harvester (Brandel, Gaithersburg, Md.) followed by three filtration washes with 200 mL of ice-cold wash buffer. |
| Affinity data for this assay | |
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