Assay Method Information | |
| In Vitro Radioligand Binding Assay |
Description: | The competition binding assay was conducted using monoclonal mouse MOR expressed in Chinese hamster ovary (CHO) cell lines. In this assay, 30 μg of membrane protein was incubated with the radioligand [3H] naloxone in the presence of different concentrations of tested compounds in TME buffer (50 mM Tris, 3 mM MgCl2, and 0.2 mM EGTA, pH 7.4) for 1.5 h at 30° C. The bound radioligand was separated by filtration using a Brandel harvester. Specific (i.e., opioid receptor-related) binding to the MOR was determined as the difference in binding obtained in the absence and presence of 5 μM of DAMGO. The IC50 values were determined and converted to Ki values using the Cheng-Prusoff equation: Ki=IC50/[1+([L*]/KD)], where [L*] is the concentration of the radioligand and KD is the KD of the radioligand was determined. |
Affinity data for this assay | |
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