| Assay Method Information | |
| | Binding Affinity Assay |
| Description: | An appropriate amount of homogenization buffer was added to the prepared membrane, and was dispersed into a suspension using a homogenizer, protein concentration was measured to be 4 mg/ml. 100 micrograms of protein were added to each well of a 96-well plate, with a volume of 90 μl. 1 μl of the compound was added to the test wells (the highest final concentration is 10 μM, diluted fourfold, across 10 concentrations), followed by 1 μl buffer to the HPE wells, and 1 μl of haloperidol (MedChemExpress, Cat #HY-14538) to the HPE wells (final concentration of 1 μM). [3H]-(+)-pentazocine (Perkin-Elmer, Cat #NET1056250UC) was added to each well (final concentration of 10 nM). The 96-well plate was incubated in a constant temperature water bath (25° C., 180 minutes). After incubation, the suspension was quickly filtered in the 96 wells through a GF/C plate prepared in advance with 0.25% PEI solution using vacuum filtration, followed by washing the GF/C three times with assay buffer. After washing, the samples were dried in a 37° C. oven. 50 μl/well of scintillation fluid (Perkin Elmer, Cat #6013621) was added to the GF/C plate. The GF/C plate was placed into a liquid scintillation counter (Perkin Elmer 1450 MicroBeta TriLux) and operated according to the program to read the experimental values. |
| Affinity data for this assay | |
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