Premium grade capillaries

The binding affinity of proteins for P ligands was determined using Microscale Thermophoresis (MST) with a Monolith NT.115 instrument (NanoTemper Technologies, Germany). Protein (100 nM) was labelled with RED-tris-NTA dye (Nanotemper Technologies) according to the manufacturer’s instructions in 50 mM HEPES pH 7.4, 250 mM NaCl, 0.05% Tween-20. A volume of 10 µl of 100 nM labelled protein was mixed with 10 µl of ligand in 50 mM HEPES pH 7.4, 250 mM NaCl, 0.05% Tween-20. 4 µl of protein–ligand mixture was loaded into Premium grade capillaries (Nanotemper Technologies) and thermophoresis was measured at 22 °C for 22 s with 20% LED power and 40% infrared laser power. Data from three independent measurements were combined and analysed using the MO.Affinity Analysis software version 2.1 (Nanotemper Technologies), fitted to a single binding site model (Eq. 1) where [l] is the concentration of ligand and data plotted using Igor Pro version 7 (Wavemetrics Inc., USA). $$f\left(l\right)\mathrm{=\; Unbound+}\frac{\left(\mathrm{Bound}-\mathrm{Unbound}\right)\times \left(\left[l\right]\mathrm{+}\left[\mathrm{protein}\right]\mathrm{+}{K}_{\mathrm{d}}-\sqrt{{\left(\left[l\right]\mathrm{+}\left[\mathrm{protein}\right]\mathrm{+}{K}_{\mathrm{d}}\right)}^2-4\mathrm{\times }\left[l\right]\mathrm{\times }\left[\mathrm{protein}\right]}\right.}{2\mathrm{\times }\left[\mathrm{protein}\right]}$$
Equation 1 shows the single binding site model used to determine dissociation constants.

20 μM protein was labeled with a two-fold excess of NT-647-NHS dye (NanoTemper Technologies, Munich Germany), following the manufacturer’s instructions. Protein had a degree of labeling of 1 dye molecule per protein molecule. Binding affinity of proteins for ligands was determined using Microscale Thermophoresis (MST) with a Monolith NT.115 instrument (NanoTemper Technologies, Germany). 10 μL of 20 nM labelled protein was mixed with 10 μL of ligand in 40 mM citrate-phosphate, 250 mM NaCl, 0.05% Tween-20 pH 7.4 or 5.4 μL of protein-ligand mixture was loaded into “Premium Grade Capillaries” (NanoTemper Technologies) and thermophoresis was measured at 22 °C for 22 s with 40% LED power and medium thermophoresis power. Data from three independent measurements were combined and analysed using the MO.Affinity Analysis software version 2.1 (NanoTemper Technologies), fitted to a single binding site model (Eq. 1) where U is the unbound normalised fluorescence, B is the fully bound fluorescence, [l] is concentration of ligand. Data were plotted using Igor Pro version 7.05 (Wavemetrics Inc., USA). $$f(l)=\mathrm{U}+\frac{(\mathrm{B}-\mathrm{U})\times [l]+[\mathrm{HtxB}]+K_{\mathrm{d}}-\sqrt{{([l]+[\mathrm{HtxB}]+K_{\mathrm{d}})}^2-4\times [l]\times [\mathrm{HtxB}]}}{2\times [\mathrm{HtxB}]}$$