Mst nt 115 device

The binding affinity of PHT1 to TASL_1-13 was measured by microscale thermophoresis using the MST NT.115 device (NanoTemper Technologies). A Cy5 dye was fused to the C-terminus of the TASL_1-13 peptide containing a polyethylene glycol (PEG) spacer. A 2-fold dilution series of PHT1 protein was prepared in 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.02% DDM/ 0.004%CHS and 5 % DMSO with the highest concentration in the assay being 28 µM. Labeled TASL_1-13 peptide at a concentration of 25 nM was mixed with the substrate dilution series and incubated for 10 min at room temperature before loading the samples in standard capillaries. For the competition assay, the same procedure was used but 120 µM of Sb27 or negative control sybody were added to each PHT1 dilution series and incubated for 10 min prior to mixing with labeled TASL peptide. Measurements were performed using 20 % LED and medium MST power. Three independent experiments were performed, yielding similar results. The data were analyzed using the MO. Affinity Analysis software (NanoTemper Technologies). Figures were prepared using Prism 9 (GraphPad).

The binding affinity of spike to Sb23 was measured by microscale thermophoresis using the MST NT.115 device (NanoTemper Technologies). Sb23 was labeled via the His₆-tag with RED-tris-NTA dye (NanoTemper Technologies) in PBS-T (10 mM phosphate, 2.7 mM KCl and 137 M NaCl, pH 7.4 supplemented with 0.05% (v/v) Tween-20), following the manufacturer’s instructions. A 2-fold dilution series of spike protein was prepared in PBS-T with the highest concentration in the assay being 1250 nM. A 50 nM final concentration of labeled Sb23 was mixed with the substrate dilution series and incubated for 10 min at room temperature before loading the samples in standard capillaries. For the competition assay, the same procedure was used but the spike dilution series were prepared in PBS-T buffer containing 800 nM of ACE2 (200 nM final concentration in the assay) and incubation of 10 min prior to mixing with labeled Sb23. All measurements were performed in triplicate using 35% LED and medium MST power. The data were analyzed using the MO. Affinity Analysis software (NanoTemper Technologies). Figures were prepared using Prism 8 (GraphPad).