Microcal peaq itc

Binding affinities of GMPPNP-bound KRAS (2–169) and KRAS (1–169) with RAF1-RBD were measured using isothermal titration calorimetry (ITC). Protein samples were prepared by extensively dialyzing them in a buffer (filtered and degassed) containing 20 mM HEPES (pH 7.3), 150 mM NaCl, 5 mM MgCl₂ and 1 mM TCEP. For the ITC experiment, 60 μM of KRAS and 600 μM of RAF1-RBD were placed in the cell and syringe, respectively. ITC experiments were performed in a MicroCal PEAQ-ITC (Malvern) at 25 °C using 19 injections of 2.2 μl injected at 150-s intervals. Data analysis was performed based on a binding model containing “one set of sites” using a nonlinear least squares algorithm incorporated in the MicroCal PEAQ-ITC analysis software (Malvern).

PCNA and peptides used in our study were extensively dialysed against PBS containing 1 mM β-mercaptoethanol. The concentration of the peptides was determined at A205 nm, using a molar absorptivity calculated specifically from the amino-acid sequence60 (link).
The measurements were performed at 25 °C using MicroCal PEAQ-ITC (Malvern). In a standard experiment, the reaction chamber was filled with a 15 μM solution of PCNA. The peptides were injected stepwise (20 injections of 2 μl). Concentrations of the peptides in the syringe were as follows: p21 (147 μM); Polι (398 μM); FEN1 (410 μM); ZRANB3 PIP (311 μM); ZRANB3 APIM (327 μM). The obtained results were processed and analysed using MicroCal PEAQ-ITC Analysis Software. The free Gibbs energy (ΔG), Enthalpy change (ΔH) and stoichiometry (N) were determined by Levenberg–Marquardt curve-fitting method employing single set of independent binding sites model. Association constant (K_A) was determined using the following equation ΔG=−RTln (K_A). Association constant (K_D) is inverse function of the dissociation constant K_A. Finally, the entropy difference (−TΔS) was obtained from the following equation: ΔG=ΔH–TΔS.

Isothermal titration calorimetry (ITC) measurements were performed with a MicroCal PEAQ-ITC (Malvern, United Kingdom) at 25°C. The sample cell was loaded with 250-μl EtoX sample (100 μM), and the reference cell contained distilled water. The syringe was filled with 75-μl ligand (PE, G1P, G3P, or PC) at a concentration of 1 mM. The EtoX protein and the four ligands were kept in the same buffer containing 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Titrations were carried out by adding 0.4 μl of substrate for the first injection and 1.5 μl for the following 14 injections, with a stirring speed of 800 rpm. The data were analyzed with MicroCal PEAQ-ITC analysis software.

Isothermal titration calorimetry (ITC) analysis was carried out at 25 °C using a MicroCal PEAQ-ITC (Malvern, Malvern, UK) to determine aptamer affinity. The binding buffer for ITC analysis contained 20 mM Tris-HCl (pH 7.5) and 20 mM NaCl. During ITC measurements, the reference power was set to 10 μcal/s and the stirring speed of the syringe was 750 rpm. Cd²⁺ solution (200 μM) from the injection syringe was titrated into the aptamer solution (20 μM) in a sample cell. After 60 s initial delay, the experiment began with the first 0.4 μL of Cd²⁺ solution and 19 successive 2.0 μL of Cd²⁺ solution every 100 s. The binding curves were obtained by integrating the heat pulse areas of each titration. Dissociation constants (K_ds), enthalpy change (ΔH), and entropy change (TΔS) were obtained by fitting the one-site binding model with the packaged MicroCal PEAQ-ITC analysis software.