Microcal peaq itc instrument

All ITC experiments were recorded using a MicroCal PEAQ ITC instrument (Malvern Panalytical, UK). Experiments were performed at 4°C and both protein and ligand were in the buffer 100 mM Tris-HCl pH 8.0, 100 mM NaCl, and 5 mM CaCl₂. The calorimetric cell was filled with 100 μM monomer concentration of either B. subtilis Noc (WT), Noc (R89A), or Noc (N121S), and was titrated with 3 mM CTPɣS. For each ITC run, a single injection of 0.5 μL of 3 mM CTPɣS or CDP was performed first, followed by 19 injections of 2 μL each. Injections were carried out at 120 s intervals with a stirring speed of 750 rpm. Each experiment was run in duplicate. The raw titration data were integrated and fitted to a one-site binding model using the built-in software of the MicroCal PEAQ ITC. Controls (CTPɣS/CDP into buffer and buffer into protein) were performed and no signal was observed.

Isothermal titration calorimetry (ITC) measurements were performed at 16 °C using a MicroCal PEAQ-ITC instrument (Malvern Panalytical). Proteins and peptides were prepared in an ITC buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 10 μM Zn²⁺). ITC experiments were performed by titrating 1.5 μL (with the exception of the first injection of 0.5 μL) of peptides (0.6–1.0 mM) into cell containing 180–220 μM proteins, with a spacing time of 90 s and a reference power of 10 μcal s⁻¹. The titration data were analyzed using the one-site binding model from MicroCal PEAQ-ITC Analysis Software version 1.30. All ITC assays were repeated at least three times independently with similar results.

ITC titrations were performed in triplicates along with control experiments (Eu(III) titration to 0.1 M NaCl) on a Malvern Panalytical, Kassel, Germany, MicroCal PEAQ-ITC instrument.
The ligand was loaded in the cell, Eu(III) in the syringe, and we added in 19 steps; the first was 0.2 µL and the following were 2.0 µL each in volume. Between injections, the system was equilibrated for 150 s. The reference cell was filled with water, the stirring speed set to 750 rpm, and the initial delay was set to 60 s.
For an accurate thermodynamic characterization of the complexes, it is useful to avoid any background reactions. As has been shown recently, many buffers show some interaction with lanthanides [57 (link)]. Therefore, work was carried out in unbuffered solutions. For EDTA and EGTA, due to the low pH values, no pH change was observed during the titration. The situation is different for NTA. The pH of the solution is lowered by the protons released during complexation. This gradual pH change was taken into account in the evaluation of the data.

Isothermal titration calorimetry (ITC) experiments were performed on a MicroCal PEAQ-ITC instrument (Malvern Ltd., Malvern, UK) at 25 °C. Protein samples and peptides were solubilized in the same buffer containing 50 mM Tris–HCl, 150 mM KCl pH 7.5 and 5 mM CaCl₂ or 5 mM EGTA. All the samples were filtered and degassed before use, and the pH was carefully determined to exclude any possible pH-related effect on ITC experiments.
A typical ITC experiment was performed by titrating 200 μL of a 30–50 μM protein solution with a 1 or 1.5 μL injection of 0.3–0.5 mM peptide (total injections 39 or 26, respectively) with an initial delay of 120 s and keeping a time gap of 120 s between injections. A reference injection of peptide into buffer without centrin was performed and subtracted from each experiment. The resulting curves were fitted to calculate apparent dissociation constants K_d, enthalpy changes (ΔH), and the apparent entropy change (ΔS) using the software MicroCal ITC Origin 7 Analysis Software (MicroCal, Malvern Ltd., Malvern, UK). All measurements were repeated in triplicate using at least two different protein preparations and the error was calculated as the mean standard error of the three measurements.

ITC was carried out on a MicroCal PEAQ-ITC instrument (Malvern Panalytical). Ligands were dissolved in DMSO as 100 mM stocks and the concentration of DMSO was then matched in the titrant and cell solutions to minimize heat changes from buffer mismatch. Experiments utilized 12 × 3.0 µl or 15 × 2.5 µl injections, with the reference response of ligand titrated into buffer being subtracted. All experiments used a cell temperature of 25°C and the data were fitted with a one-site model using the manufacturer’s software.

hsDNA was dialyzed overnight against 150 × 10⁻³m KCl, 10 × 10⁻³m N‐2‐hydroxyethylpiperazine‐N'‐2‐ethanesulfonic acid (HEPES) (pH 7.4 containing 0.5% DMSO) at 4 °C. The compound Cor‐gal was dissolved to 5 × 10⁻⁶ and 10 × 10⁻⁶m in the same buffer used for DNA. Solutions were degassed by ultrasonic device and centrifuged at 15 000 × g for 10 min before titration. Then Cor‐gal solution was added into the sample cell and the DNA solution was loaded in the syringe for titration. The ITC experiment was performed with a MicroCal PEAQ‐ITC instrument (Malvern Instruments, Great Britain) at 25 °C at the time intervals of 150 s and a stirring speed of 750 rpm. 100 × 10⁻⁶m hsDNA was conducted by 13 aliquots and titrated into the calorimetry cell containing either Cor‐gal or control buffer. The first injection of 0.4 µL DNA was ignored to eliminate the diffusion effects from the syringe on the sample cell, and the following injections were carried out at 3 µL. The cell filled with dialyzed buffer titrated by DNA was assigned as control to get the blank data. Final data were analyzed with the MicroCal PEAQ‐ITC analysis software. The “one set of binding sites” fitting model was applied to analyze the thermodynamic parameters.