| Assay Method Information | |
| | Competitive Kappa Receptor Binding Assay |
| Description: | Step 1: 50 μL of a vehicle (1% DMSO) was added into a total binding tube (TB), 50 μL of U69593 (with a final concentration of 1.0×10−5 M) was added into a non-specific binding tube (NB), and 50 μL of a test compound was added into each compound binding tube (CB).Step 2: 100 μL of a buffer (homogenate A) was added into each reaction tube.Step 3: the prepared membrane was suspended in the homogenate A to obtain a 15 mg/mL membrane suspension for later use.Step 4: 50 μL of radioligand 3H-U69593 was added into each reaction tube to reach a final concentration of 2 nM.Step 5: 50 μL of the membrane solution was added into each reaction tube.Step 6: each reaction tube was incubated at 25° C. for 90 min. After the reaction was completed, the binding ligands were rapidly filtered under reduced pressure. The UniFilter GF/C plate was saturated with 0.5% PEI solution 1 h in advance, fully washed with cold Tris buffer, filtered under vacuum, then placed into a thermostatic dryer, and dried for 30 min. The filter plate was taken out, and MICROSCINT PS scintillation solution was added at 40 μL/well.Step 7: the filter plate was put into a liquid scintillation counter for counting. |
| Affinity data for this assay | |
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