| Assay Method Information | |
| | ThermoFluor (Tf) assay |
| Description: | The ThermoFluor is a fluorescence based assay (Tf) that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., et al., J. Biomol. Screen 2001, 6, 429-40.). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).In a ThermoFluor experiment where protein stability is monitored as the temperature is steadily increased, an equilibrium binding ligand causes the midpoint of an unfolding transition (Tm) to occur at a higher temperature. The shift in the melting point described as a ΔTm is proportional to the concentration and affinity of the ligand. The compound potency may be compared as a rank order of either ΔTm values at a single compound concentration or in terms of KD values, estimated from concentration response curves.The details of the KEAP1 KELCH ThermoFluor Assay Construct are as follows: Kelch domain of human KEAP1 (321-624 aa) was used in the assay. The protein was expressed in E. coli with 6His tag that was cleaved prior to receipt for use.ThermoFluor experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein (KEAP Kelch) and compound solutions were dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Reference wells contained KEAP Kelch without compounds, and the assay conditions were as follows: 1.1 μM (0.037 mg/mL) KEAP Kelch, 80 μM 1,8-ANS, 25 mM PIPES, pH 7.0, 100 mM NaCl, 0.002% Tween-20.The binding affinity was estimated as described previously (Matulis, D. et al., Biochemistry 2005, 44, 5258-66) using thermodynamic parameters of protein unfolding listed below. |
| Affinity data for this assay | |
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