Tryptophan fluorescence quenching assay

Intrinsic tryptophan fluorescence quenching assays were performed in black greiner 96 well plates using the PHERAstar FSX plate reader at 28°C with the optical model FL ex280 em340. The final protein concentration was 1 μM and the ligand concentration 0–50 μM. The sample volume was 120 μl/well; the ligand stocks were made in DMSO and the reactions were performed in 20 mM Tris pH 7.5, 200 mM NaCl, 0.5 mM EDTA 0.02% DDM, 0.004% CHS. Samples were incubated at room temperature for 10 min before the measurement. All measurements were performed in triplicates. Apparent K_d values were calculated using non-linear regression in GraphPad Prism 7.0.