| Assay Method Information | |
| | Determination of Affinity for Human 5-HT2A Receptors in Competitive Binding Assay |
| Description: | Receptor membranes were prepared from the CHO-K1 recombinant AequoScreen® cell line stably expressing the human 5-HT2A receptor (PerkinElmer, Waltham, MA, USA). Cells were suspended in 4× volume in buffer A (15 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA) (1 g cell-4 mL buffer) and homogenized in a Dounce homogenizer. The crude membrane fraction was collected following two consecutive centrifugation steps at 40,000×g for 25 minutes separated by a washing step in buffer A. The final pellet was resuspended in buffer B (75 mM Tris-HCl, pH 7.5, 12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose) in a concentration of 80 mg wet cell weight in 0.5 mL buffer, aliquoted and flash frozen on dry ice. Protein content was determined using the bicinchoninic acid assay in the presence of sulfhydryl reagents with bovine serum albumin (BSA) as a standard. |
| Affinity data for this assay | |
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