Measurements were made using a MicroCal AutoITC200 (Malvern, United Kingdom). Purified CAMKK2-KD was dialyzed into gel filtration buffer (20 mM Hepes, pH 7.5, 300 mM NaCl, 5% glycerol, and 1 mM TCEP - overnight at 4 °C). Small molecule inhibitors were diluted in dialysis buffer. All ITC experiments were performed using a “reverse titration” setup in which the protein (in the syringe) was titrated into the cell containing the inhibitor. Two independent ITC measurements were performed for each compound, data for individual runs were integrated separately to obtain single isotherms, which were plotted and analyzed using Sedphat37 (link). For BI 2536, CAMKK2-KD was used at 106.8 µM (run 1) and at 130 µM (run 2); the inhibitor was used at 10.7 µM (run 1) and at 13 µM (run 2). For ALK-IN-1, CAMKK2-KD was used at 105.6 µM (run 1) or at 106.6 µM M (run 2); the inhibitor was used at 10.6 µM (run 1) and at 10.7 µM (run 2). For GSK650394, CAMKK2-KD was used at 130 µM (run 1) or at 105.8 µM (run 2); the inhibitor was used at 13 µM (run 1) and at 10.6 µM (run 2). For CP-673451, CAMKK2-KD was used at 105.8 µM and the inhibitor was used at 10.6 µM in both runs. All measurements were made at 20 °C with mixing (1,000 rpm stirring), and used 1.5 µL injections and 180 s between each injection. NITPIC, SEDPHAT and GUSSI37 (link) were used to analyze and generate figures for ITC data.
Auto itc200
Manufactured by Malvern Panalytical
Sourced in United Kingdom, United States
The Auto-iTC200 is a fully automated isothermal titration calorimetry (ITC) system designed for high-throughput analysis of biomolecular interactions. The instrument can perform automated liquid handling, temperature control, and data analysis, enabling efficient characterization of binding affinities, thermodynamics, and stoichiometry.
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Lab products found in correlation
Origin 7, by OriginLab (14 mentions)
Origin 7, by Malvern Panalytical (4 mentions)
Itc200, by Malvern Panalytical (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Origin software, by Malvern Panalytical (3 mentions)
Microcal itc200, by Malvern Panalytical (3 mentions)
Origin version 7, by Malvern Panalytical (2 mentions)
Cp225d, by Sartorius (2 mentions)
Peaq analysis software, by Malvern Panalytical (2 mentions)
Itc origin 7.0 analysis software, by Malvern Panalytical (2 mentions)
Microcal peaq itc analysis software 1, by Malvern Panalytical (2 mentions)
Nano itc, by TA Instruments (1 mentions)
Microcal origin, by GE Healthcare (1 mentions)
Microcal origin 7, by Malvern Panalytical (1 mentions)
M3895, by Merck Group (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Origin 8, by Malvern Panalytical (1 mentions)
Origin version 7, by OriginLab (1 mentions)
Peaq itc analysis software, by Malvern Panalytical (1 mentions)
Cary 100 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
80 protocols using auto itc200
1
Isothermal Titration Calorimetry of CAMKK2-KD Inhibitors
2
ITC Analysis of PRC2-Compound Interactions
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ITC experiments were performed on an Auto ITC200 (Microcal) at 25°C. The ITC sample cell was filled with 10 μM PRC2 in titration buffer containing 25 mM HEPES pH8.0, 150 mM NaCl, and 1% DMSO. 40 μL of 100 μM compounds in the same titration buffer was loaded in the ITC syringe. 19 injections, each of 2 μL, was injected into the ITC cell at 150 s intervals. Binding constant (K) and binding enthalpy (ΔH) determinations were obtained from the fit to the experimental data using a one-site model.
3
Thermodynamic Analysis of scFv-CBZ-GA-pyridine
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The thermodynamic parameters of the interactions between scFv and CBZ-GA-pyridine were measured with a MicroCal Auto-iTC200 (Malvern, UK). A 50 µM aliquot of the CBZ-GA-pyridine was titrated with 5 µM of scFv in HBS-EP buffer at 25 °C. The titration curves were analyzed by using MicroCal Origin Software (Malvern, Malvern, UK). The measurement at 37 °C was performed by using Nano ITC (TA Instruments, New Castle, DE, USA). The dissociation constants and thermodynamic parameters were calculated by the one set of sites fitting model.
4
Calorimetric Titration of PADI4 and NLS1/2-PADI4 Peptides with Importins
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Calorimetric titrations for testing the interaction of PADI4, as well as for the interaction of NLS1/2-PADI4 peptides, with both importins, Impα3 and ΔImpα3, were carried out in an Auto-iTC200 automated high-sensitivity calorimeter (MicroCal, Malvern-Panalytical, Malvern, UK). Experiments were performed in 20 mM Tris buffer (pH 7.5), 5 mM TCEP, 150 mM NaCl, and 5% glycerol at 25 °C. PADI4 or the peptide solution (100 μM) in the injection syringe was titrated into the importin solution (10 μM) in the calorimetric cell. The remaining experimental and processing details have been described previously [41 (link),42 (link),43 (link)]. Due to the presence of glycerol in solution, background injection (included as an adjustable parameter in data fitting) was rather large. The data analysis was conducted in Origin 7.0 (OriginLab, Northampton, MA, USA) with user-defined fitting functions.
5
Binding Affinity of XfMqsR-XfYgiT Interaction
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The isothermal titration calorimetry (ITC) experiments were performed at 25°C using an Auto iTC200 calorimeter (MicroCal, Northampton, MA, USA). To determine the binding constant and the heat of the interaction, the recombinant proteins were extensively dialyzed against sodium phosphate buffer (25 mmol.L−1sodium phosphate at pH 7.8, 150 mmol.L−1NaCl, and 0.3 mmol.L−1TCEP) at 25°C. Thirty-three injections of 1 μL of XfMqsR at 100 μmol.L−1 were titrated into ~204 μL of XfYgiT at 10 μmol.L−1, with 300 s intervals between each injection.
The obtained heat signals from the raw ITC data were integrated using Origin software (MicroCal Inc., Northampton, MA). The heat from the reference buffer and the titrant, which is the heat due to the diffusion of the protein into the buffer, was subtracted to calculate the corrected heat release. A single-site binding isotherm model was used to match the data and to assess the dissociation affinity (Kd), the enthalpy (ΔH), the entropy (ΔS), and the binding stoichiometry (N).
The obtained heat signals from the raw ITC data were integrated using Origin software (MicroCal Inc., Northampton, MA). The heat from the reference buffer and the titrant, which is the heat due to the diffusion of the protein into the buffer, was subtracted to calculate the corrected heat release. A single-site binding isotherm model was used to match the data and to assess the dissociation affinity (Kd), the enthalpy (ΔH), the entropy (ΔS), and the binding stoichiometry (N).
6
Isothermal Titration Calorimetry of HsrA-DHP Interactions
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Isothermal titration calorimetry (ITC) experiments were carried out in an Auto-iTC200 calorimeter (MicroCal, Malvern-Panalytical, Malvern, United Kingdom) in order to determine several thermodynamic parameters of the molecular interaction between HsrA and selected DHP derivatives. The protein, located in the calorimetric cell at 20 μM, was titrated with each ligand, located in the injection syringe at 200 μM, by programming a series of 19 injections of 2 μl, with 150 s time spacing, 10 μcal/s reference power, and 750 rpm stirring speed (Velázquez-Campoy et al., 2015 (link)). Experiments were performed in buffer 50 mM Tris–HCl (pH 8), 150 mM NaCl, 10% glycerol, 1% DMSO at two different temperatures (15°C and 25°C) in order to obtain the best signal. The heat effect per injection was normalized by the amount of ligand injected, and the interaction isotherm was analyzed by nonlinear least-squares regression data analysis considering an interaction model with a single ligand binding site in the protein, using user-defined fitting routines in Origin 7.0 (OriginLab, Northampton, MA, United States).
7
Isothermal Titration Calorimetry of Ligand-Protein Binding
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Varying concentrations of ligands and proteins (100 µM) were solubilized in 50mM Tris pH 8.0, 150mM NaCl, 0.5mM TCEP. Calorimetry experiments were performed using a microcalorimeter (iTC200, MicroCal) at 24 °C, by titration of the ligand (1 × 0.5 µL + 16 × 2.5 µL injections at 280 s intervals; stirring speed of 1000 rpm). Titrations of ligands into buffer were performed as a control, and the resulting heats of ligand dilution were subtracted from the experimental data prior to curve fitting. MicroCal Data Analysis software, Auto-iTC200 was used to monitor ligand binding behavior using non-linear least-squares fitting assuming a single-site model.
8
Isothermal Titration Calorimetry of Peptide-Protein Interactions
9
Isothermal Titration Calorimetry of DmpR
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The ITC experiments were conducted using a MicroCal Auto-iTC200 at 25 °C at the Korea Basic Science Institute (KBSI). The DmpR solution (10 µM or 40 µM) in the calorimetric cell was titrated with the phenol ligand (100 µM), cognate DNA with specific UAS sequences (100 µM), or σ54(1–119)-CPD protein (400 µM) as the injectant. The data were analysed with the MicroCal Origin software package (GE Healthcare).
10
Divalent Cation Binding to YqgP Domains
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The binding of divalent cations to the extramembrane domains of YqgP (NTD, CTD) was investigated using titration microcalorimetry. All titration experiments were performed in 50 mM Tris–HCl, pH 7.3, 150 mM NaCl at 25°C using Auto‐iTC200 instrumentation (MicroCal, Malvern Panalytical Ltd). Prior to the ITC measurements, protein samples were dialysed into the same buffer in which divalent cation salt solutions were prepared. Typically, 200 μl of the protein sample was titrated by stepwise 2 μl injections of divalent cation solution. Protein and ligand concentrations were adjusted as follows: 1.5 mM NTD was titrated by 50 mM MnCl2, 50 mM MgSO4, 50 mM ZnCl2 or 50 mM CaCl2, 0.7 mM NTD by 15 mM CoCl2 and 0.25 mM NTD by 7 mM NiCl2‐buffered solutions. Control experiments included ligand titration into the buffer only. Concentrations of stock solutions of the analysed proteins were determined by quantitative amino acid analysis, and concentrations of the stock solutions of divalent metal cation salts were determined by elemental analysis. Titration data were processed and analysed using MicroCal Origin 7.0 (MicroCal, Malvern Panalytical Ltd).
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