Fluorescent Labeling and Binding Assay

The purified TLR4 ectodomain and soluble CD14 were labelled with DyLight-488. The labelling reaction was performed according to the manufacturer’s instructions. Full-length TLR4, CD14 and MD2 were obtained from HEK293 lysates. The GFP-tagged TLR4, CD14 and MD2 constructs were transfected into HEK293, incubated for 48 h and lysed in RIPA buffer and clear supernatant was obtained by the centrifugation.
The labelled TLR4 or CD14 was adjusted to 20 nM with MST buffer, and the lysates were diluted according to their fluorescence intensity. Recombinant CnB was dissolved in MST buffer to the appropriate concentration. A series of 16 1:1 dilutions were prepared, and the labelled proteins or GFP-tagged receptors from the lysates were added to each ligand dilution and mixed. After 10-min incubation, each solution was added to Standard Treated Capillaries (NanoTemper Technologies). Thermophoresis was measured using a Monolith NT.115 instrument (NanoTemper Technologies) at an ambient temperature of 25 °C with 5 s/30 s/5 s laser off/on/off times, respectively. The instrument parameters were adjusted to 50% LED power and 20% MST power. The data from three independently pipetted measurements were analysed (NT.Analysis software version 1.5.41, NanoTemper Technologies) using the signal from Thermophoresis + T-Jump36 (link)37 (link).