Mst grade glass capillaries

Purified YfiB wild-type and it mutant YfiB^L43P were fluorescently labeled using the NanoTemper blue protein-labeling kit according to the manufacturer’s protocol. This resulted in coupling of the fluorescent dye NT-495. PG was titrated in 1:1 dilutions starting at 1 mmol/L. To determine of the K_d values, 10 μL labeled protein was mixed with 10 μL PG at various concentrations in Hepes buffer (20 mmol/L Hepes, 200 mmol/L NaCl, 0.005% Tween-20, pH 7.5). After 10 min of incubation, all binding reaction mixtures were loaded into the MST-grade glass capillaries (NanoTemper Technologies), and thermophoresis was measured with a NanoTemper Monolith-NT115 system (20% light-emitting diode, 20% IR laser power).

MST experiments were performed on a Monolith NT.115 instrument (NanoTemper Technologies). Proteins were labelled with the red fluorescent dye NT-647 using the Protein Labeling Kit RED-NHS (NanoTemper Technologies). The concentration of the labelled protein was kept constant at 100 nM, while the concentration of the compound was varied. A 15-step twofold dilution series beginning at 500, 250 or 125 µM finally yielded 16 different concentrations of the tested compound [(1)–(4)]. Experiments were carried out in 10 mM HEPES buffer pH 8 containing 100 mM NaCl, 1 mM DTT, 0.05%(w/v) Tween 20, 0.25 mM MgCl₂ and 0.25 mM MnCl₂. The final samples were adjusted to 5% DMSO to ensure the solubility of the compounds. The samples were centrifuged for 5 min at 13 000 rev min⁻¹ to remove potential aggregates and the supernatant was loaded into standard treated MST-grade glass capillaries (NanoTemper Technologies). After a 5 min incubation period the MST was measured with 80% LED power and 80% infra-red laser power. K_d values were determined using the NanoTemper analysis software.