Igor pro version 7

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.

The colorimetric variety of the new ALucs with various CTZ analogs was investigated in MDA cells (Figure 3).
MDA cells containing the new ALucs or Rluc8.6-535 were prepared via lipofection using the same method used in Figure 1D. The cells were passively lysed with a lysis buffer (Promega) according to the manufacturer’s instructions. The lysates, 40 μL each, were aliquoted into 200 μL PCR tubes, and each tube was injected with 40 μL of PBS, dissolving each substrate. The tube was immediately mounted in the sample stage of a spectrophotometer (AB-1850, ATTO, Tokyo, Japan), and the corresponding BL spectra were determined and analyzed using the specific software LumiFLSpectroCapture version 1.0 and IGOR Pro version 7 (WaveMetrics, Portland, OR, USA).