Assay Method Information

Assay Name:  Ligand Binding Assay
Description:  Human RORγ ligand binding assay was performed in 96-well format. The N-terminal DNA binding domains (DBD) of the native RORγ and RORγt receptors have been substituted with that of the yeast GAL4-DBD and RORγ is expressed constitutively in the cell line. Both agonist and inverse agonist activity can be detected. 10 mM compound stocks were diluted serially 1:3 with DMSO and further diluted with provided media to generate 10 titration points from 60 μM to 3 nM. These treatment conditions were added to the plates as 2× media in 100 μL volume. Each plate includes a positive control with 10 titration points as well as 6 negative control wells with vehicle only. Reporter cells were rapidly thawed and added to the plates in 100 μL volume. The plates were incubated for 24 hrs in a 37° C. humidified 5% CO2 incubator. Media was removed before the addition of room temperature Detection Substrate. After 5 minute incubation, the luminescence was quantified on a luminescence plate reader.
Affinity data for this assay
 

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