Prometheus nt plex nanodsf instrument

Cyani HAP2 ectodomain purified from Expi293F cells (20 mg/mL in 1 μL 20 mM Tris-HCl, pH 7.5, 500 mM NaCl was diluted with 19 μL of buffers containing 500 mM NaCl and the following: 50 mM HCI-KCI (pH 1.5 and pH 2.0), citric acid-trisodium citrate (pH 3.5, pH 4.3, and pH 5.0), or Tris-HCl (pH 7.5). Duplicate samples in nanoDSF Grade Standard Capillaries (NanoTemper Technologies) were subjected to thermal unfolding in a Prometheus NT.Plex nanoDSF instrument (NanoTemper Technologies) with a linear thermal ramp of 2 °C/min from 20 °C to 95 °C with excitation at 275 nm at a power of 30%. The fluorescence intensity ratio (FIR) of tryptophan emission at 350 nm/330 nm was determined.

Differential scanning fluorimetry (DSF) was carried out to determine the thermal stability of the Mef1 protein. The concentration of Mef1 (5 µM) was determined by measuring the absorbance at 280 nm using a calculated molar extinction coefficient computed using ProtParam from ExPASy. Samples were added to 10 µl capillary tubes and analyzed using a Prometheus NT.Plex nanoDSF instrument (Nanotemper Technologies, Munich, Germany). Thermal stability was monitored using a temperature gradient of 25–80°C with an increase of 1°C min⁻¹ to obtain denaturation profiles. Raw data were exported into data sets that contained fluorescence at 330 and 350 nm (F₃₃₀ and F₃₅₀) as well as the ratio of these values (F₃₃₀/F₃₅₀) and absorbance at 350 nm (A₃₅₀). The first derivatives of F₃₃₀/F₃₅₀ were plotted to visualize denaturation. The peak of the first derivative gives the value of the melting temperature (T_m), at which half of the protein is unfolded.

Differential scanning fluorimetry (DSF) was carried out to determine the thermal stability of the Mef1 protein. The concentration of Mef1 (5 µM) was determined by measuring the absorbance at 280 nm using a calculated molar extinction coefficient computed using ProtParam from ExPASy. Samples were added to 10 µl capillary tubes and analyzed using a Prometheus NT.Plex nanoDSF instrument (Nanotemper Technologies, Munich, Germany). Thermal stability was monitored using a temperature gradient of 25–80°C with an increase of 1°C min⁻¹ to obtain denaturation profiles. Raw data were exported into data sets that contained fluorescence at 330 and 350 nm (F₃₃₀ and F₃₅₀) as well as the ratio of these values (F₃₃₀/F₃₅₀) and absorbance at 350 nm (A₃₅₀). The first derivatives of F₃₃₀/F₃₅₀ were plotted to visualize denaturation. The peak of the first derivative gives the value of the melting temperature (T_m), at which half of the protein is unfolded.