| Assay Method Information | |
| | Radioligand Binding Assay |
| Description: | Radioligand dose displacement assays used 0.4-0.8nM [3H]-U69,593 (NEN; 40 Ci/mmole) with 10-20 ug membrane protein (recombinant kappa opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 uL binding buffer (5% DMSO, 50mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 10 uM unlabeled naloxone or U69,593. All reactions were performed in 96-well polypropylene plates for 1 h at a temperature of about 25C. Binding reactions were determined by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Packard) followed by five filtration washes with 200uL ice-cold binding buffer. Filter plates were subsequently dried at 50C for 1-2 hours. Fifty uL/well scintillation cocktail (MicroScint20, Packard) was added and plates were counted in a Packard Top-Count for 1 min/well. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |