ITC experiments were performed at 30°C using an ITC-200 microcalorimeter from Microcal (GE Healthcare) following the standard procedure as reported previously [34] (link). Proteins were prepared in 100 mM MES, pH 6.0, 200 mM KCl. In each titration, 20 injections of 2 µL each of SGTA_NT (dimer), at a concentration of 500 µM, were added to a sample of BAG6_UBL or UBL4A_UBL respectively at 50 µM (monomer). Integrated heat data obtained for the titrations corrected for heats of dilution were fitted using a nonlinear least-squares minimization algorithm to a theoretical titration curve, using the MicroCal-Origin 7.0 software package. ΔH (reaction enthalpy change in Kcal/mol), Kb (equilibrium binding constant in per molar), and n (molar ratio between the proteins in the complex) were the fitting parameters. The reaction entropy, ΔS, was calculated using the relationships ΔG = −RT lnKb (R = 8.314 J/(mol K), T 303 K) and ΔG = ΔH−TΔS. Dissociation constants (Kd) are shown for each interaction.
Itc200 microcalorimeter
Manufactured by GE Healthcare
Sourced in United States
The ITC200 microcalorimeter is a calorimetry instrument designed to measure the heat changes associated with molecular interactions. It provides accurate and sensitive measurements of the thermodynamic parameters of biomolecular binding events.
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Lab products found in correlation
Origin 7, by OriginLab (6 mentions)
600 and 700 mhz spectrometers, by Bruker (2 mentions)
800 and 600 mhz, by Bruker (2 mentions)
Origin software version 7, by Malvern Panalytical (1 mentions)
Microcal origin 7, by Malvern Panalytical (1 mentions)
Origin 7, by Malvern Panalytical (1 mentions)
Membrane filter, by Merck Group (1 mentions)
Origin software, by GE Healthcare (1 mentions)
63 protocols using itc200 microcalorimeter
1
Thermodynamic Analysis of BAG6-SGTA Interaction
2
Thermodynamic Characterization of RNA-Ligand Interactions
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ITC titrations were performed at 298 K using an ITC-200 microcalorimeter (GE Healthcare). RNA solutions (30–60 µM) were prepared by diluting concentrated stocks into the binding buffer containing 40 mM HEPES (pH 7.2), 100 mM KCl, 10 mM MgCl2. Guanidine and diguanidine compounds were prepared in the same binding buffer with a concentration of 0.5–1 mM. Solutions were degassed for 2–5 min before loading. The sample cell was filled with 200 µL of RNA. Guanidine or diguanidine was injected in a volume of 0.4 µL for the first injection and 2 μL for the next 19 injections using a computer-controlled 40 µL microsyringe with an injection interval of 120 sec. Titration of ligands into the binding buffer or titration of the binding buffer into the RNA solution resulted in negligible evolution of heat. Integrated heat data were analyzed using a one-set-of-sites model in MicroCal Origin following the manufacturer's instructions. The first data point was excluded in analysis. The binding parameters ΔH (reaction enthalpy change in cal mol−1), K (binding constant in M−1), and n (bound ligands per RNA) were variables in the fit. The binding free energy ΔG and reaction entropy ΔS were calculated using the relationships ΔG = −RT ln K, where R = 1.987 cal mol−1 K−1, T = 298 K and ΔG = ΔH − TΔS. The dissociation constant Kd was calculated as 1/K.
3
Isothermal Titration Calorimetry of PCNA
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All ITC experiments were carried out at 25°C on an iTC200 Microcalorimeter (GE Healthcare) with a 200 μl cell capacity and 40 μl syringe volume. A degassed 50 mM NaCl, 25 mM Tris–Cl pH 7.4 buffer was used for all solutions and for the control measurements. Twenty injections (starting with a 0.4 μl pre-injection followed by 19 injections of 2 μl) with a spacing of 150 s and stirring speed of 800 rpm were carried out. Concentrations ranged from 1.5–2 mM for the peptides and 150–200 μM for the purified PCNA constructs. All measurements were carried out at T = 298.15 K; the control injections were subtracted from sample measurements to correct for the heat during dilution. The data points were analyzed with the MicroCal Origin 7.0 software using a one-site binding model. The total heat exchanged with PCNA during each injection of PARG peptides was fitted to a single site binding model and the fitted curve was used to extract the thermodynamic properties. The summary of thermodynamic data is provided in Supplementary Table S3 .
4
Calcium-Dependent Binding of SOAR and CaM
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Measurements were performed at 16 °C using an ITC-200 microcalorimeter (GE Healthcare). Samples were buffered with 25 mM Tris-HCl (pH 7.5) containing 200 mM NaCl and 2 mM CaCl2 or 1 mM EGTA. To determine the binding affinity of SOAR and CaM, 500 μM CaM or CaM-4EF was injected into 50 μM SOAR. Data were analyzed in the Microcal Origin software.
5
Quantifying ParE-CopA Binding Affinity
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ITC was applied to quantitatively determine the binding affinity of the ParESO-CopASO complex or CopASO to a 25 bp DNA duplex derived from the copASO-parESO promoter (5′-TAAGGTA TTAC CTAGTA GTAC TAAG-3′; the palindromic sequences are underlined). For the titration experiments, the protein was purified using the same method as above and was dialyzed against the buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl, and 5% (v/v) glycerol for 24 h. DNA was dissolved in the same buffer as above. The ITC experiments were carried out using a high-sensitivity iTC-200 microcalorimeter from Microcal (GE Healthcare) at 20 °C using 100–400 μM DNA in the injector with 10–100 μM ParESO-CopASO complex or CopASO in the sample cell. All samples were thoroughly degassed and then centrifuged to get rid of precipitates. Injection volumes of 2 µL per injection were used for the different experiments, and for every experiment, the heat of dilution for each ligand was measured and subtracted from the calorimetric titration experimental runs for the protein. Consecutive injections were separated by 2 min to allow the peak to return to the baseline. Integrated heat data obtained for the ITCs were fitted in a one-site model using a nonlinear least-squares minimization algorithm to a theoretical titration curve, using the MicroCal-Origin 7.0 software package.
6
Isothermal Titration Calorimetry of Peptide Interactions
7
Isothermal Titration Calorimetry of PGP
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Purified murine PGP-WT or PGP-P127E was dialyzed overnight against ITC buffer (30 mM HEPES, 30 mM NaCl, 5 mM MgCl2, 1 mM tris(2-carboxyethyl)phosphine/TCEP; pH 7.5). All calorimetric measurements were performed at 25 °C on an ITC200 microcalorimeter (GE Healthcare). Experiments were conducted with a fixed concentration of 2% DMSO. The heat signals released by 15 consecutive 2.4 μL injections of 1 mM CP1 or 0.5 mM CP3 solutions that were titrated into 100 μM or 50 μM PGP, respectively, were measured under continuous mixing at 750 rpm. Both CP1 and CP3 were diluted from a 50 mM stock solution in 100% DMSO, and PGP solutions likewise contained 2% DMSO. To reduce the effects of ligand leakage from the syringe during baseline equilibration, the heat release of a single 1.2 μL injection was recorded prior to the actual titration experiment, and discarded during data analysis with Nitpic 2.0.745 (link). To determine background heats of dilution, control experiments of ligand into buffer, buffer into protein and buffer into buffer titrations were performed.
8
VIN3VEL Monomer Affinity Determination
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To determine the affinity between VIN3VEL monomers, ITC was carried out at 25°C with an iTC 200 Microcalorimeter (GE Healthcare). Titrations consisted of 19 consecutive 2 μL injections of 1 mM 6xHisLip-VIN3VEL I575D (following a pre-injection of 0.5 μL) into 100 μM 6xHisLip-VIN3VEL RR>AD at time intervals of 180s with constant stirring at 750 rpm, in 25 mM Tris pH 7.4, 200 mM NaCl, 0.5 mM DTT and 0.06% NaN3, and the data were analyzed using MicroCal PEAQ-ITC Analysis Software (1.1.0.1262, Malvern Sciences).
9
Thermodynamic Characterization of HrBP1-Q-R Binding
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At 25°C, ITC-based binding experiments were performed using an ITC 200 Micro Calorimeter (GE Healthcare). The HrBP1 protein was in the buffer containing 20-mM Tris-HCl and 100-mM sodium chloride (pH 7.5). The 2 mM Q-R compound was titrated into the HrBP1 protein (0.1 mM) in a 200-μL sample cell as follows: 0.4 μL for the first injection and 2 μL for the next 19 injections using a 40-μL microsyringe, the intervals of each injection was150 s. According to the manufacturer’s instructions, the integrated heat data were analyzed using the one-set-of-sites model. The first data point was not required in the analysis. The binding parameters reaction including enthalpy change, ΔH (cal⋅mol–1), binding constant K (mol–1) and number of molecules per HrBP1 protein (n) was floating in the fit. The binding free energy and reaction entropy was calculated (Kumar et al., 2014 ). The dissociation constant Kd was determined as 1/K (Kumar et al., 2014 ).
10
Characterizing Peptide-Protein Interactions
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All samples were exchanged into fresh buffer (20 mM Tris-HCl, 150 mM NaCl and 1 mM TCEP). ITC measurements were performed at 20°C on an ITC200 microcalorimeter (GE Healthcare). In each titration, the protein in the cell (at a 10–30 μM concentration) was titrated with 25 × 4 μl injections (at 180 s intervals) of protein ligand (at 10-fold higher molar concentration). The following synthetic peptides (purity >95%, Genscript) were used Thr11 DST(p)PRTLLRRVLDTAYA, Thr85 EQT(p)PRTLLKNILLTAYA, Thr27 PRT(p)PRRPRSARAGARYA, and Thr47 TAS(p)PRKLSGQTRTIARYA. Injections were continued beyond saturation to allow for determination of heats of ligand dilution. Data were fitted by least-square procedure to a single-site binding model using ORIGIN software package (MicroCal).
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