| Assay Method Information | |
| | Binding Assay |
| Description: | Binding to the CB1R was assessed in a competition displacement assays using [3H]CP-55,940 as the radioligand and crude membranes from mouse brain for the CB1R, as reported previously. Membranes from cells expressing human CB2R were purchased from Charles River, (Cat #A308; Ohio, US). Solutions of test compounds ranging from 0.1 nM to 10 mM were prepared in DMSO. The desired amount of membrane preparation was diluted with ice-cold assay buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2, 0.1% BSA, pH 7.4) and was vortexed. 100 μL of compound was distributed into each tube, followed by addition of 800 μL of diluted membranes (1 μg/tube) and kept on ice until the addition of [3H]CP-55,940. [3H]CP-55,940 was diluted with cold (unlabeled) assay buffer and 100 μL was added into each tube. The assays were incubated for 90 minutes at 30° C. and then immediately filtered on WHATMAN GF/B FilterPaper (Fired) using a Brandel M-24R Harvester followed by six washes with ice cold wash buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2, 0.1% BSA, pH 7.4). Radioactivity was detected by adding the FilterPaper directly to the ULTIMA GOLD scintillation cocktail (PerkinElmer), incubation at 20° C. for 60 min and then counted using a Tri-Carb 4910TR liquid scintillation counter. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |