| Assay Method Information | |
| | Radioligand Binding Assay |
| Description: | Plasma membranes prepared from HEK 293 EBNA cells transfected with human CB1 or CB2 cannabinoid receptors (3.7 or 3.3 .mu.mol/mg protein, receptor concentration; PerkinElmer) were used for the radioligand binding studies. The composition of incubation buffer for CB1 assay was 50 mM Tris base, 2.5 mM EDTA, 5 mM MgCl.sub.2 and 0.5 mg/ml fatty acid free BSA and for CB2 assay was 50 mM Tris base, 2.5 mM EGTA, 5 mM MgCl.sub.2 and 1 mg/ml fatty acid free BSA; pH was adjusted to 7.4 by adding 1N HCl. Plasma membranes were diluted with incubation buffer to provide a final protein concentration of 2.4 .mu.g (CB1) or 8 .mu.g (CB2) per well in non-binding surface polystyrene 96-well assay plates (Corning). Compound solutions were prepared in silanized glass tubes and dispensed using pipette tips with SUPERSLIK.TM. surface. Competition studies were performed using a final concentration of 0.72 nM [.sup.3H]-CP 55,940 (100-180 Ci/mmol, specific activity; PerkinElmer) against test compounds. |
| Affinity data for this assay | |
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