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| | [35S] GTPgammaS binding test |
| Description: | In a [3SS] GTPγS binding test, the opioid receptor agonist activity of test compounds based on a GTP-GDP exchange reaction was evaluated. In the test, a cell membrane sample (75 μg/well) was seeded in a 96-well microplate, and test compounds at various concentrations, 30 μM guanosine-5′-diphosphate (GDP: Sigma-Aldrich Co., MO. USA) and 100 pM [35S] GTPγS (PerkinElmer Co., Ltd.) were added thereto and the incubation was carried out at 300 rpm at 25° C. for 2 hours. After the incubation was completed, filtration was performed with a Filtermat B glass filter (PerkinElmer Co., Ltd.) which had been previously soaked in 50 mM tris-HCl (pH 7.4) at 4° C. using a FilterMate cell harvester (PerkinElmer Co., Ltd.). Following filtration, the glass filter was washed three times with 50 mM tris-HCl (pH 7.4), and then the filter was dried at 60° C. for 90 minutes using a dryer. After drying, Meltilex B/HS (Perkin Elmer Co., Ltd.) was melted and allowed to infiltrate into the glass filter on a hot plate at 90° C., and the glass filter was put into a clear film case and the case was loaded into the measuring cassette of a Microbeta 2 (PerkinElmer Co., Ltd.). The radioactivity on the glass filter was measured with the Microbeta 2 (PerkinElmer Co., Ltd.), and nonspecific binding was calculated from the difference in binding capacity between in the presence and absence of a nonradioactive ligand (10 μM GTPγS: Sigma-AldricH Co.). The 50% effective concentration (EC50 value) of the test compound obtained by the [35S] GTPγS binding test was calculated from the S-shaped curve obtained by the test using GraphPad Prism 6 (GraphPad Software, Inc., CA, USA). |
| Affinity data for this assay | |
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