| Assay Method Information | |
| | Surface Plasmon Resonance |
| Description: | Binding assay to Kelch domain of KEAP1 was performed by Surface Plasmon Resonance (SPR) on a Biacore T200 (GE Healthcare) at 25° C. using CM5 chip. His-Thromb-KEAP1(A321-C624) acquired from Novalix (batch PU-P01) was immobilized covalently by amine coupling using standard wizard protocol, at a concentration of 20 μg/mL in sodium acetate pH 5.5. The ligand was captured until 1250 RU was reached. Amine blank is used as the reference flow cell. Binding of analyte to the ligand was monitored in real time with associations of 180 seconds and a dissociation of 720 seconds. Association (ka) and dissociation (kd) rates were fitted with a simple 1:1 kinetic interaction model using Biacore T200 Evaluation Software (GE Healthcare). The equilibrium constant (KD=kd/ka) was calculated from these fitted parameters. In the experiment, a half-log dilution of the compound was injected over KEAP1 in Single Cycle Kinetics (SCK) with 5 increasing concentrations of 0.316, 1, 3.16, 10 and 31.6 nM per cycle. The Biacore binding assay was performed with a flowrate of 50 μL/min using a running buffer of 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 1 mM TCEP, 0.05% P20,5% DMSO. Double subtraction was used by subtracting both reference flow cell and previous blank cycle (with running buffer). Solvent correction was applied to correct DMSO bulk effect. |
| Affinity data for this assay | |
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