Compounds are typically subjected to primary functional screening assay at targets (receptors) selected by investigators. In primary screening assays, compounds are tested in quadruplicate at a final concentration of 10 μM or at specific concentrations upon request for agonist and antagonist activities. Results are normalized and transformed to percentage values. For agonists, the reference agonist activity at 10 μM is set as 100% and basal activity with buffer as 0%. For antagonists, the basal activity with buffer only is set as 100% inhibition and the activity of the EC80 of the reference agonist as 0% inhibition. For allosteric potentiators, the activity of the EC20 of a reference agonist is set as 0% potentiation. Compounds with a minimum of 30% agonist activity, or 50% antagonist activity, or 30% potentiation above control are selected for secondary screening using concetnration-response assays.
In secondary functional assays, potential agonist hits are subjected to full concentration-response (8 concentrations in quadruplicate) assay to determine their efficacy and potency. Potential antagonist hits are subjected to full concentration-response (8 concentrations in quadruplicate) assay to determine IC50 values against EC50to EC80 concentrations of a reference agonist. Potential antagonist hits and positive allosteric modulators are further characterized if necessary.
Both primary and secondary functional binding assays are carried out in a final of volume of 125 μl per well in appropriate binding buffer. [35S]-GTPγS is at a concentration specific for the target receptor (unless stated otherwise). Basal and nonspecific binding are determined in the absence and presence of 10 μM of GTPγS. Agonist & Antagonist activity is determined by receptor specific compounds.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 buffer 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 functional 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.