Evaluation of TR-FRET assay performance

Robustness of the assay was evaluated through the determination of the Z' factor. The potent inhibitor 2 (1 μM in 10 mM HEPES pH 7.4 buffer containing 1% DMSO, final concentration) and blank 10 mM HEPES pH 7.4 buffer containing 1% DMSO were used as the positive and negative controls, respectively. A pre-incubated mixture (10 μL) of His-tagged Keap1 Kelch domain protein (final, 5 nM) and Tb-anti-His antibody (final, 0.5 nM) was dispensed into wells of a 384-well assay plate already containing 0.2 μL of the inhibitor solution or neat DMSO (33 replicates each). After a 30-min incubation, FITC-9mer Nrf2 peptide amide (final, 25 nM) was added. The assay mixture was further incubated for 1 h before the TR-FRET signals were measured. The Z' factor was calculated using the following equation:

Where SDp and SDn are the standard deviation of the positive and negative control wells, respectively; whereas μp and μn are the mean TR-FRET signal values of the positive and negative controls, respectively.