Table 2.

Summary of Glucocorticoid Receptor Competitor Assay

Step	Parameter	Value	Description
1	Compound addition to plate	0.5 μL	1 or 4 mM compound in DMSO added to black 384-well plates
2	Buffer addition	19.5 μL	1×GR screening buffer, 1×Stabilizing peptide, 5 mM DTT
3	Fluorescent ligand addition	10 μL	4×Fluormone™ in assay buffer
4	Protein addition	10 μL	4× GR in assay buffer
5	Incubation time	2 h	Covered with aluminum foil at ambient temperature
6	Assay readout	485 and 513 nm	Fluorescence polarization, 100 readings/well

1. 1 or 4 mM compound used for 12.5 and 50 μM final concentrations, respectively.

2. Room temperature assay performed.

4. Prepare GR on ice.

 DTT, dithiothreitol.