Compounds are typically subjected to primary radioligand binding assays at targets (receptors) selected by investigators. In the primary binding assays, compounds are usually tested at single concentrations (10 μM) in quadruplicate in 96-well plates. Compounds that show a minimum of 50% inhibition at 10 μM are tagged for secondary radioligand binding assays to determine equilibrium binding affinity at specific targets.
Both primary and secondary radioligand binding assays are carried out in a final of volume of 125 μl per well in appropriate binding buffer. The hot ligand concentration is usually at a concentration close to the Kd (unless stated otherwise). Total and nonspecific binding are determined in the absence and presence of 10 μM of the appropriate reference compound, respectively. In brief, plates are incubated at a specific temperature in the dark for a specific amount of time (receptor specific).
Reactions are stopped by vacuum filtration onto 0.3% polyethyleneimine (PEI) soaked 96-well Unifilters using a Filtermate harvester, followed by a wash with cold wash buffer. Scintillation cocktail is then added onto air-dried filters. Radioactivity is counted in a Microbeta counter.
Key details of our binding assay services:
Gold standard filtration assays.
Over 100 targets covered.
Single concentration screening or dose-response for IC50 determinations.
Percent inhibition (mean of replicates).
Individual values as a percent of control, IC50 value (calculated from a minimum of 6 concentration testing).
Plotted IC50 curves.
Proven skills and expertise to develop difficult targets.
Reliable, quick turnaround times.
High quality data with strict quality controls.
Flexible custom services to supplement our off-the-shelf services to meet your needs, providing a cost-effective way to expand your capabilities and capacity.