| Assay Method Information | |
| | Affinity of Compounds for Dopamine D2 Receptors |
| Description: | The affinity of the compounds of the present disclosure for the dopamine D2 receptors was determined by the method of radioligand competition experiment. In the first step, a cell membrane component containing specific dopamine D2 receptors was prepared. A 10 cm culture dish was used for transfection with 10 ng of the dopamine D2 receptors and 40 μL of polyethylenimine (PEI hereafter). After 48 hours, the 10 cm culture dish was taken out from the cell culture incubator and the cultured cells had expressed the dopamine D2 receptors. A vacuum pump was used to suck off the culture medium, 3 mL of lysis buffer was added to each culture dish, and the cells were placed in a 4° C. cold room for 10 minutes. After the cells were detached, the cells were transferred to a 15 mL centrifuge tube and centrifuged at 1500 rpm for 5 minutes at 4° C., and the supernatant was discarded. The cell pellet was transferred to a tissue homogenizer, and 3 mL of lysis buffer was added and fully ground until the cells were broken. Then, cell suspension was equally aliquoted into several EP tubes, centrifuged at 12000 rpm for 5 minutes at 4° C., and the supernatant was discarded. The precipitate was the cell membrane component containing the dopamine D2 receptors. In the second step, a ligand-receptor binding experiment was performed on 293T membrane component transiently expressing the dopamine D2 receptors. First, a standard binding buffer was added to the cell membrane component containing the dopamine D2 receptors, and the cell membrane was disrupted and resuspended with an electric tissue homogenizer. 30 μL of membrane protein suspension was added to each well of a 96-well plate. |
| Affinity data for this assay | |
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