ITC measurements were performed on an iTC200 instrument (GE Healthcare) in duplicate. 500 µm of rCEACAM-N domains were loaded into the syringe of the calorimeter and 50 µm rR28-IgI3 was loaded into the syringe. All measurements were performed at a stirring speed of 750 rpm, in 30 mm Tris-HCl, 150 mm NaCl, pH 7.5 at 25 °C. Origin 7.0 software was used to analyze all data.
Itc200 instrument
Manufactured by GE Healthcare
The ITC200 instrument is a calorimetry device designed for thermodynamic analysis. It measures the heat released or absorbed during chemical or biological processes, providing data on the energetics of molecular interactions.
Automatically generated - may contain errors
Lab products found in correlation
Origin software, by GE Healthcare (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Origin software, by OriginLab (1 mentions)
Vp itc, by GE Healthcare (1 mentions)
Origin v7, by Malvern Panalytical (1 mentions)
Origin 7, by GE Healthcare (1 mentions)
Uv vis spectrophotometer, by Beckman Coulter (1 mentions)
14 protocols using itc200 instrument
1
Calorimetric Analysis of CEACAM-N:R28-IgI3
2
Isothermal Titration Calorimetry of Kinase Interactions
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All ITC experiments were performed on itc200 instrument (GE Healthcare). Prior to ITC titration, proteins and peptides were buffer exchanged into 10 mM HEPES pH 7.4, 500 mM NaCl, 0.05% P20 surfactant. ITC experiments with Aurora Awt kinase were performed in buffer containing 50 mM HEPES pH 7.4, 100 mM Mg(CH3COO)2, 100 mM NaCl, 1 mM ATP, 1 mM DTT, 0.01% v/v P20. Titrations experiments were conducted at 25°C with an initial 0.4 μl injection at a duration of 0.8 s, followed by 19 or 29 injections of 2 μl at a duration of 4 s with 120, 180 or 200 s spacing for Aurora AD274N, Aurora AWT and CK2α kinase binding assays, respectively. In the Aurora AD274N and Aurora AWT binding assays, 50 μM solution of the titrant was injected into 5 μM kinase solution in the cell. For the determination of binding affinity between CK2α and its interacting partners, 100 μM of CK2β peptide or RAD-CK2β display were titrated into 10 μM CK2α kinase solution. Binding isotherms were fit by non-linear regression using the single-site model provided by ORIGIN software (Origin Lab). The stoichiometry of the interaction (N), equilibrium association constant (KA) and change of enthalpy (ΔH) were floated during the fitting.
3
Isothermal Titration Calorimetry of Protein-RNA Binding
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Isothermal titration calorimetry (ITC) experiments were performed using iTC200 instrument (GE) at 25°C. The protein and the RNA samples were thoroughly de-gassed before the experiment. For the titrations, 100–200 μM of the protein was titrated into 5 μM of the RNA filled in the sample cell. 20 injections containing 2 μl of the titrant each were made keeping an interval of 180 s between each injection. The integrated heat data for all the runs was corrected for the heat of dilution, the protein being injected at each step. The data was then fitted for one site binding model using ORIGIN software provided by the vendor (GE). All the parameters were kept floating during the data fitting. All the experiments were repeated at least two times for data consistency.
4
Quantifying BauA Binding to Iron-Siderophore Complexes
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Affinity of BauA for Fe3+-preacinetobactin and Fe3+-acinetobactin were measured by isothermal titration calorimetry using a VP-ITC or a ITC200 instrument (GE Healthcare) at 25 ˚C. Ratio 1:2 and 1:1 of Fe3+: preacinetobactin or acinetobactin have been tested. Solutions of 100 mM of compound and either 100 mM or 50 mM of Fe3+-acetylacetonate in DMSO were diluted with the dialysis buffer (50 mM Sodium phosphate pH 7.5, 50 mM NaCl, 0.8% OctylPOE). Titrations of preacinetobaction were performed using 2 μl injections of 100 μM of Fe3+ and either 100 μM or 200 μM preacinetobactin into 10 μM BauA.
Several conditions have been tested for Fe3+-acinetobactin. Titrations of acinetobaction were performed using 2 μl injections of 100 μM of Fe3+ and 200 μM acinetobactin (1:2) into 10 μM BauA. Titrations of acinetobaction were also performed with higher concentration using 5 μl injections of 370 μM of Fe3+-acinetobactin (1:1) in 20 μM BauA in the same buffer. The heats of dilution were measured by injecting the ligands into the buffer. Titration curves were fitted using Origin software.
Several conditions have been tested for Fe3+-acinetobactin. Titrations of acinetobaction were performed using 2 μl injections of 100 μM of Fe3+ and 200 μM acinetobactin (1:2) into 10 μM BauA. Titrations of acinetobaction were also performed with higher concentration using 5 μl injections of 370 μM of Fe3+-acinetobactin (1:1) in 20 μM BauA in the same buffer. The heats of dilution were measured by injecting the ligands into the buffer. Titration curves were fitted using Origin software.
5
Isothermal Calorimetry Study of SH3-FUS Interactions
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Proteins were dialyzed in the same buffer (150 mm KCl, 10 mm imidazole (pH 7.0), 1 mm EGTA, 1 mm MgCl2, and 1 mm tris(2-carboxyethyl)phosphine) overnight before isothermal calorimetry measurements. Their concentrations were determined by absorbance at 280 nm with an Agilent 8453 UV-visible spectrometer. Measurements were performed at 20 °C on an iTC200 instrument from GE Healthcare. 20 μm SH33, SH33–FUS(27S), or SH33–FUS(27L) (molecule concentration) was loaded into the cell, and 200 μm PRM4 (molecule concentration) was loaded in the syringe and titrated into the cell. Each injection contained 2 μl PRM4. The time interval between injections was 120 s so that the system could come to equilibrium after each injection. Isotherms were generated using the NITPIC software.
6
Isothermal Titration Calorimetry of DNA-Protein Interactions
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Isothermal titration calorimetry (ITC) experiments were performed using iTC200 instrument (GE) at 25°C. Samples were thoroughly degassed before the experiment. For the titrations, the sample cell was filled with 5 μM of the DNA and titrated with 100–200 μM of the protein. 20 injections of the titrant were performed at an interval of 3 min between each injection. The heat of dilution was subtracted from the integrated heat data and the data was fit for one site binding model using ORIGIN software provided by the vendor (GE). All the parameters were kept floating during the data fitting.
7
Characterizing CaV1.1 Peptide Binding
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All proteins were concentrated and dialyzed against 500 mM NaCl, 20 mM Hepes pH 7.4, and 2 mM TCEP at 4 °C. The synthetic CaV1.1 peptide was dissolved in the dialysis buffer. Protein concentrations were determined with the Edelhoch method (63 (link)). Titrations consisted of 20 injections of 2 µL with concentrations noted in the figure legends. Experiments were performed at 25 °C and using a stirring speed of 750 rpm on an ITC200 instrument (GE Healthcare). All data were processed by using Origin (v7.0), and isotherms were generated by following a point-by-point subtraction of a reference titration of ligand into buffer.
8
Calmodulin-Ligand Binding Kinetics
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The calmodulin lobes were dialyzed
overnight against 150 mM KCl, 10 mM Na-Hepes pH 7.4, 2 mM CaCl2. The Eu3+ complexed sevoflurane analogue and control
ligand were dissolved into the same buffer at a final concentration
of 10 mM. Titrations consisted of 20 injections of 2 μL ligand
at 10 mM into the cell containing 1 mM calmodulin lobe. The background
heats from dilution of the ligands were determined by titrating them
into buffer. Experiments were performed at 25 °C and a stirring
speed of 750 rpm on an ITC200 instrument (GE Healthcare). The data
were processed using Origin 7.0 and fit to a single-site fitting model
after background buffer subtraction.
overnight against 150 mM KCl, 10 mM Na-Hepes pH 7.4, 2 mM CaCl2. The Eu3+ complexed sevoflurane analogue and control
ligand were dissolved into the same buffer at a final concentration
of 10 mM. Titrations consisted of 20 injections of 2 μL ligand
at 10 mM into the cell containing 1 mM calmodulin lobe. The background
heats from dilution of the ligands were determined by titrating them
into buffer. Experiments were performed at 25 °C and a stirring
speed of 750 rpm on an ITC200 instrument (GE Healthcare). The data
were processed using Origin 7.0 and fit to a single-site fitting model
after background buffer subtraction.
9
Quantifying Ubiquitin Binding to USP47
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Ubiquitin-binding to USP47 was performed using the ITC200 instrument (GE Healthcare) at 25 °C, and the data were analyzed using the program ORIGIN 7.0. Prior to titration, the protein samples were centrifuged at 18,188×g at 4 °C for 5 min to remove any debris. Experiments were conducted by titrating 500 µM (except for the mutants of cUSP47CDH178F and cUSP47CDH178A using 1 mM) monoubiquitin into the cell containing ~25–30 µM of prepared wild type and each mutant of USP47 protein in identical buffer with injectant. All proteins were prepared by dialyzing in a buffer of 20 mM HEPES (pH 7.4), and 100 mM NaCl at 25 °C. The injections were 2 μL each, with an injection interval of 150 s. The stoichiometry (n), association constant (KD), and the change in enthalpy (ΔH), were obtained by using a nonlinear least-squares curve-fitting algorithm.
10
Isothermal Titration Calorimetry of Ligand Binding
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ITC measurements were performed with an iTC200 instrument (GE Healthcare) at 25 °C. All the proteins and ligands (DMXAA, CMA, 2′3′-cGAMP, 3′3′-cGAMP) were in buffer C (30 mM HEPES, pH7.5, 150 mM NaCl). A typical titration experiment involved 19 injections of ligand (2 mM) solution into the ITC cell containing protein (0.2 mM). The data were fitted to titration curves with Origin v7.0 (MicroCal).
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