| Assay Method Information | |
| | Radioligand Competition Binding Assay |
| Description: | For all experiments, serotonin (5-HT) hydrochloride was used as a positive control, and an appropriate high affinity, cold ligand (e.g., mianserin hydrochloride, 10 μM, for 5-HT2Rs) was used to define non-specific binding. Each cell membrane homogenate expressing a 5-HTR was incubated at room temperature in 96-well plates with [3H]lysergic acid diethylamide ([3H]LSD) or [3H]ketanserin between 0.5 and 1 nM or [3H]mesulergine at appr. 3 nM for labeling 5-HT2Rs, or [3H]5-carboxamidotryptine (5-CT) between 0.2 and 1 nM for labeling 5-HT1A,1B,1DRs in the absence or presence of test articles (e.g., at 10 concentrations from 0.3 nM to 10 μM in half-log units) in a buffer for 90 min. Following equilibration, each sample was filtered rapidly under vacuum through fiberglass filters presoaked in a buffer and washed by vacuum several times with an ice-cold buffer. Filters were soaked with scintillation fluid and counts per minute were detected using photodetectors (Microbeta Scintillation Counter).. The Kd for [3H]LSD was set to 0.7-1.0 nM (depending on the 5-HT2R subtype labeled), for [3H]ketanserin was set to 1.6 nM for 5-HT2A, for [3H]mesulergine was set to 2.92 nM for 5-HT2C, for [3H]5-CT was set to 0.2-2.0 nM (depending on the 5-HT1R subtype labeled), and the IC50 values were computed using nonlinear, least squares regression analyses and then converted to binding affinity (Ki) values using the Cheng-Prusoff equation (GraphPad Prism 9.0, San Diego, California). Data shown are the results from all experiments combined. As is known, G protein-coupled receptors (GPCR), including the 5-HT2AR, can exist in multiple conformations, and ligands, typically agonist ligands, have unique affinities for these conformations of the receptor. |
| Affinity data for this assay | |
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