Prometheus nt 48 high sensitivity capillaries

Thermal stability of SPM proteins was performed by nano-differential scanning fluorimetry (nanoDSF) using a Prometheus NT.48 instrument (NanoTemper Technologies, Munich, Germany). Ca-SPM and Ca-SPM-P415A were dialyzed overnight at 4°C against TN buffer, and the freshly prepared protein samples were supplemented with increasing concentrations of calcium ions (0 to 10 mM CaCl₂) before 10 μl was used for filling the Prometheus NT.48 high-sensitivity capillaries (NanoTemper Technologies). The measurements were conducted from 20 to 95°C (with a temperature ramp of 2.5°C/min) under constant monitoring of tryptophan fluorescence at 350 and 330 nm. The melting temperature (T_m) values, corresponding to the inflection points of the unfolding curve, were determined via the first derivative of the curve.

PBRM1-BD4 proteins were prepared at a concentration of 30 μM in buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 10% v/v glycerol, 1 mM tris(2-carboxyethyl)phosphine) for a final volume of 40 μl. Samples were drawn up in triplicate in Prometheus NT.48 high-sensitivity capillaries (NanoTemper Technologies, PR-C006) and run at 100% excitation power with a temperature ramp of from 20 °C to 95 °C increasing 1 °C/min in a Prometheus NT.48 instrument (PR001). As PBRM1-BD4 lacks Trp residues, intrinsic Tyr fluorescence at 330 nm was plotted versus temperature, and PBRM1-BD4 protein T_m was determined by identifying the maximum of the first derivative of the fluorescence signal with ThermControl software version 2.3.1 (NanoTemper Technologies, https://nanotempertech.com/prometheus/nt48-software).

Two-dimensional denaturation
of dEngBF proteins was followed using
the Prometheus NT.48 (NanoTemper) recording fluorescence emission
at 330 and 350 nm upon excitation at 280 nm. A denaturation dilution
series was prepared by mixing different volumes of protein sample
adjusted to a concentration of 15 μM with and without guanidine
hydrochloride. The samples were loaded into Prometheus NT.48 high
sensitivity capillaries (NanoTemper technologies cat. no. PR-C006)
and sealed with high vacuum grease (Dow Corning) to avoid evaporation
during the experiment. The excitation power was set to ensure that
the fluorescence signal was within the linear range of the instrument.
The temperature range was 20–95 °C with a temperature
increment of 1 °C/min. For each candidate, the melting curves
for each concentration of denaturant were fitted to the following eq 3 correcting for pre- and
post-transition by adding quadratic terms:^{29 (link)} where is the change in enthalpy upon unfolding
at T_m, ΔC_p^o is the change in the heat capacity of
the system assumed to be independent of temperature.^{30 (link)}T_m is the melting temperature
and R is the gas constant while the constants a₀, a₁, and b₁ are constants describing the temperature dependence
of the fluorescence signal. ΔT is defined as
ΔT = T – T_ref where T_ref is an arbitrary
reference temperature.