Assay Report Card

ID: ALA3705238

Type: Binding

Description: Radioligand Binding Assay: Membranes for radioligand binding experiments are prepared from fresh or frozen cells as described in Klotz et al., Naunyn-Schmiedeberg's Arch. Pharmacol, 357:1-9 (1998). The cell suspension is then homogenized in ice-cold hypotonic buffer (5 mM Tris/HCl, 2 mM EDTA, pH 7.4) and the homogenate is spun for 10 minutes (4° C.) at 1,000 g. The membranes are then sedimented from the supernatant for 30 minutes at 100,000 g and resuspended in 50 mM Tris/HCl buffer pH 7.4 (for A3 adenosine receptors: 50 mM Tris/HCl, 10 mM MgCl2, 1 mM EDTA, pH 8.25), frozen in liquid nitrogen at a protein concentration of 1-3 mg/mL and stored at −80° C. Dissociation constants of unlabeled compounds (Ki-values) are determined in competition experiments in 96-well microplates using the A1 selective agonist 2-chloro-N6-[3H]cyclopentyladenosine ([3H]CCPA, 1 nM) for the characterization of A1 receptor binding. Nonspecific binding is determined in the presence of 100 μM R-PM and 1 mM theophylline, respectively.

Organism: Homo sapiens