| Assay Method Information | |
| | competitive radioligand displacement and functional assays |
| Description: | The affinity of ligands was determined via radioligand binding techniques using human recombinant receptors expressed in mammalian clonal cells. Details on assay conditions, radioligand, nonspecific binding and receptor expression are shown in Table 5. Number of independent radioligand binding experiments is shown in Table 6. In Table 6, all independent experiments listed were performed using 3 technical replicates.Ligand affinity was assessed using established methodology and 2-5 pg of protein per well, determined by the Pierce bicinchoninic acid protein assay according to the manufacturers protocol (Thermo Scientific), (Perry, et al., 2020; Roth, 2013). Saturation binding experiments were performed on membranes expressing 5-HT2A, 5-HT2B, 5-HT2C, or H1Rs in triplicate across 8 concentrations. Competition binding assays were performed in at least triplicate with approximate Kd concentrations of radioligand. Total and nonspecific binding were determined in octet. Each compound was assessed in at least two independent experiments across I Q-14 concentrations in half-log units (1 μM-100 μM) where the center of the concentration range was the approximate pKi. Serial dilutions of unlabeled compound were made in assay buffer at 2.5x the final concentration using 10 mM DMSO stocks (final [DMSO] < 1%). Assays were terminated using rapid filtration via Whatman GF/B Fired Filters (Brandel Inc., Gaithersburg,MD) soaked in 0.3% (w/v) PEI, using an automated Tomtec Harvester 96 (Hamden, CT). Filters were washed with 50 mM Tri-HCI (pH=7.4, 4 °C) before being oven dried and placed into scintillation vials containing 1 ml SX18-4 ScintiVerse BD Cocktail (Fisher Chemical, Fair Lawn, NJ). Scintillation was detected using a Tri-Carb 2910 TR Liquid Scintillation Analyzer (Perkin Elmer, Boston, MA).Table 5. Radioligand binding assay conditions for membranes used in this study, including the binding parameters used to derive K, values and the results of saturation binding assays. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |