Nano itc calorimeter

ITC (isothermal titration calorimetry) studies were carried out at 20 °C using Nano ITC calorimeter (TA Instruments, New Castle, DE, USA). The reference cell was filled with deionized water. The reaction cell contained 1000 μL of 97 μM PA in 10 mM HEPES-KOH pH 8.1 buffer. The PA solution was titrated by 2.52 μL aliquots of 3.6 mM SrCl₂ in the same buffer: 37 injections with a 200 s interval between the injections. The injection syringe was rotated at 280 rpm during the titration and pre-equilibration. The ITC data were fitted by the sequential metal binding scheme (1) using FluoTitr v.1.42 software (IBI RAS, Pushchino, Russia), implementing the nonlinear regression algorithm by Marquardt [30 (link)]. K_Me1, K_Me2 and specific enthalpies of the possible protein states were used as fitting parameters.

Calorimetric experiments were performed on a NanoITC calorimeter (TA Instruments) at 25 °C while stirring at 250 r.p.m. All proteins were buffer exchanged by gel filtration into 50 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM DTT, except for SMARCA2 and SMARCA4A BDs, which were exchanged into 10 mM Hepes pH 7.5, 500 mM NaCl and 5% glycerol. Typically, 10–100 μM compound and 60–900 μM protein were placed in the cell and syringe, respectively. Titrations consisted of an initial injection of 1.5 μl followed by 19 identical injections of 2.5 μl made at time intervals of 5 min. ITC data were corrected for the heat of dilution of injectant into buffer and analysed with software provided by the manufacturer using a single binding site model. The first data point was excluded from the analysis. Thermodynamic parameters determined from the ITC data are summarized in Supplementary Table 1.

ITC measurements were performed using a nano ITC calorimeter (TA Instruments) at 12 °C or 25 °C. Protein samples were prepared in 20 mM Tris-HCl pH 7.0 and 130 mM NaCl. All samples were degassed and centrifuged prior to the experiments. Protein concentration was measured in a NanoDrop™ 2000 measuring the UV absorption at 280 nm. Peptide concentration was determined by amino acid analysis. Depending on the expected affinity, sigmoidal curves were optimized by injecting 10- to 15-fold concentrated peptide in 16 × 3 μL steps in a cell containing 190 μL of protein at an adjusted concentration of between 150 and 200 μM , stirring at 200 revolutions per minute (rpm). The delay between injections was set to be of 3 minutes. The NanoAnalyze software (TA Instruments) was used to determine the binding isotherms, assuming a single binding site in each molecule (except for the Pin1 full-length interaction with the CPEB1 pS210 peptide, where one Pin1 molecule could interact with two peptides). Baseline controls were acquired with buffer and pure peptide solutions. Measurements were repeated at least twice.

ITC measurements were performed using a NanoITC calorimeter (TA Instruments) at 25°C. Membrane vesicles containing CbrT (200 μl, 10 mg*ml⁻¹ in 50 mM KPi, pH 7.5) were added to the NanoITC cell. Ligands were prepared in 50 mM KPi, pH 7.5 and titrated into the cell in 1 μl injections with 140 s between each injection. Membrane vesicles containing the full-complex ECF-CbrT that does not bind CN-Cbl (10 mg*ml⁻¹ in 50 mM Kpi, pH 7.5) were used as a negative control. Data were analyzed with the Nano Analyze Software.

The isothermal titration calorimetry experiments were conducted using a Nano-ITC calorimeter (TA Instruments). A 500 μM GL1 peptide solution and a 10 μM siRNA solution were both prepared in RNase-free water. All of the samples were degassed in a degassing station (TA Instruments) prior to the experiments. RNase-free water was placed in the ITC reference cell. For each titration, 2 μl of the peptide in a pipette rotating at 250 rpm was injected into the siRNA solution in the sample cell of the calorimeter, which was equilibrated to 25°C, with an interval of 300 s between injections. The heat of dilution was measured by titrating the GL1 solution into RNase-free water and was later subtracted from the sample measurement. The data were analyzed using NanoAnalyze software v.3.1.2.

ITC experiments were carried out at 20 °C using a nano-ITC calorimeter (TA Instruments). Prior to ITC analysis, both GST-RCC1 and ΔIBB-importin αs were dialyzed overnight against Gel Filtration (GF) buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM BME, 0.1 mM PMSF) at 4 °C. GST-RCC1 (300 µM) was injected in 2.0 µl increments into a calorimetric cell containing 195 µl of ΔIBB-importin α3 or α1 (100 µM). The spacing between injections was 180 s. Titrations were performed in triplicate and data were analyzed using the NanoAnalyze data analysis software (TA Instruments). Heats of dilution were determined from control experiments carried out by injecting GF buffer against importin α3/α1 and subtracted from enthalpies obtained by titrating RCC1 against importin α3/α1. Curve fitting was done in NanoAnalyze data analysis software using a single set of binding sites model. The concentration of samples used for ITC was accurately determined using amino acid analysis, Lowry assay and spectrophotometric determination with the theoretical extinction coefficient ε. In both titrations in Fig. 1, the N-value at the midpoint point is <1, as expected for a 1:1 interaction, because the active fraction of recombinant importin α isoforms in cell is likely <1.