ITC measurements were performed at 25 °C on an iTC-200 calorimeter (MicroCal, Inc). The titrations were carried out in buffer A. The reactant (0.1 mM protein) was placed in the 200-μl sample cell. Then dsDNA solutions in an injection syringe (0.6 mM) were injected into protein solutions in the cell. The volume of each injection was 2 μl except for the first injection, which was 0.4 μl. A titration experiment consisted of 20 consecutive injections of 4 s duration, with a 120 s interval between injections. Control experiments were performed under identical conditions to determine the heat signals that arise from addition of DNA into the buffer. The resulting data were fitted to a single-site binding model using the Origin software package (MicroCal, Inc).
Origin software package
Manufactured by Malvern Panalytical
The Origin software package is a data analysis and visualization tool designed for use with Malvern Panalytical's analytical instruments. It provides users with the ability to process, analyze, and present data collected from various Malvern Panalytical products.
Automatically generated - may contain errors
Lab products found in correlation
Itc200 calorimeter, by Malvern Panalytical (5 mentions)
Vp itc microcalorimeter, by Malvern Panalytical (2 mentions)
Vp itc calorimeter, by Malvern Panalytical (2 mentions)
Vp dsc system, by Malvern Panalytical (2 mentions)
Nucleotide stocks, by Merck Group (2 mentions)
Pd 10 buffer exchange column, by GE Healthcare (2 mentions)
Vp itc instrument, by Malvern Panalytical (2 mentions)
Vp isothermal titration calorimeter, by Malvern Panalytical (1 mentions)
Itc200 microcalorimeter, by Malvern Panalytical (1 mentions)
Itc200 calorimeter, by GE Healthcare (1 mentions)
Microcal peaq itc, by Malvern Panalytical (1 mentions)
Itc200, by Malvern Panalytical (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
20 protocols using origin software package
1
Protein-DNA Binding Affinity Determination
2
Quantifying Protein-Protein Interactions via ITC
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The protein-protein interactions between TrxA (or its active site mutants), MogR, and GmaR were measured using isothermal titration calorimetry (ITC) with a VP-isothermal titration calorimeter from Microcal, Inc. (Northampton, USA). Calorimetric titrations of TrxA (80–200 μM in the syringe) and PrfA, MogR, or GmaR (8–20 μM in the cuvette) were carried out at 25°C in 25 mM Tris-HCl, pH 7.5, 100 mM NaCl. TrxA or its active site mutants (C28A, C31A, and C28AC31A) was titrated into PrfA, MogR, or GmaR in 15 μL injections with a spacing of 300 s between injections. Calorimetric data were analyzed by integrating heat effects normalized to the amount of injected protein and curve-fitting based on a 1:1 binding model using the Origin software package (Microcal). The dissociation constant was derived from data using standard procedures. ITC was carried out in the Life Sciences Institute of Zhejiang University.
3
Thermodynamic Characterization of Protein-Peptide Interactions
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For the ITC measurement, the concentrated proteins were diluted into 20 mM Tris, pH 7.5, 150 mM NaCl; the lyophilized peptides (Peptide 2.0 Inc.) were dissolved in the same buffer, and the pH value was adjusted by adding NaOH. Peptides concentrations were estimated from the mass. All the measurements were performed in duplicate at 25 °C, using a VP-ITC microcalorimeter or iTC-200 microcalorimeter (MicroCal, Inc.). The protein with a concentration of 50–100 μM was placed in the cell chamber, and the peptides with a concentration of 1–2 mM in syringe was injected in 25 (19 for iTC-200) successive injections with a spacing of 180 s (150 s for iTC-200) and a reference power of 13 μcal s−1 (6 μcal s−1 for iTC-200). Control experiments were performed under identical conditions to determine the heat signals that arise from injection of the peptides into the buffer. Data were fitted using the single-site binding model within the Origin software package (MicroCal, Inc.). iTC-200 data should be consistent with those from VP-ITC instrument, based on ITC results of same PHD domain using the two instruments.
4
ITC Analysis of VP24 Interactions
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The dissociation constant (Kd) and stoichiometry of the interaction between VP24 and VP28 or VP26 were measured by ITC using an ITC200 calorimeter (GE Healthcare). Calorimetric titration of VP28 (0.3 mM in the syringe; 2 μl injections) or VP26 (0.2 mM in the syringe; 2 μl injections) to VP24 (0.012 mM or 0.03 mM in the cell, 200 μl) was performed at 25 °C in assay buffer containing 25 mM Tris-HCl, pH7.0, 200 mM NaCl, 5% glycerol or buffer containing 25 mM Tris-HCl, pH7.0, 100 mM NaCl. Time between injections was 150 s. ITC data were analyzed by integrating the heat effects after the data were normalized to the amount of injected protein. Data fitting was conducted to determine the dissociation constant and stoichiometry based on a single-site binding model using the Origin software package (MicroCal).
5
Isothermal Titration Calorimetry of STING Binding
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Isothermal titration calorimetry (ITC) was employed to measure the binding affinities between STINGR232/CTD and the purified CDNs using a MICROCAL PEAQ-ITC (Malvern Instruments, United Kingdom) (Zhang et al., 2013 (link); Du and Su, 2017 (link)). The purified STINGR232/CTD was thawed and diluted to 100 μM in a buffer containing 25 mM HEPES pH 7.8 and 150 mM NaCl. Next, it was loaded into the sample cell. A 50 mM stock solution of the CDN was prepared in pure water, diluted to 1 mM (0.8 mM for 3′3′-cGAMP) with the same buffer as the STINGR232/CTD, and loaded into the syringe injector. Titrations were performed at 25°C and involved 19 injections (1 × 0.4 and 18 × 2 μl) at 150 s intervals. A reference titration of ligand into buffer was used to correct for heat of dilution. Data fitting was based on a single-site binding model using the Origin software package (MicroCal). The dissociation constant was derived from the data by using standard procedures.
6
Thermodynamic Analysis of PurF Binding
7
Compound Binding Assay for LMPTP
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Experiments were performed at 23°C using an ITC200 calorimeter (Microcal) in a buffer containing 10 mM Tris pH 7.5, 75 mM NaCl, 0.5 mM β-mercaptoethanol and 5% DMSO. Aliquots (2 μl) of 0.5 mM compound were injected into the cell containing 50 μM LMPTP in the presence or absence of 200 μM sodium orthovanadate. Experimental data were analyzed using the Origin software package (Microcal).
8
ITC Measurement of p62 PH-D Binding
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The binding dissociation constant (Kd) for the interaction between p62 PH-D and human RPB6 (residues 1−24) or yeast (Saccharomyces cerevisiae) Rpb6 (residues 11−34) was measured by ITC using a VP-ITC calorimeter (MicroCal). Titration of 100∼300 μM RPB6/Rpb6 in the syringe (25 × 20 μl injections) into 2 ml of 10–30 μM p62 PH-D in the cell was carried out in 20 mM potassium phosphate (pH 6.8) with or without 25 mM NaCl at 20°C. Each injection took 4 s, with a pre-injection delay of 210 s and a syringe stirring speed of 307 rpm. Data were analyzed by using the Origin software package (MicroCal).
9
Differential Scanning Calorimetry of Fab Thermal Stability
10
Differential Scanning Calorimetry of Fabs
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The heat capacity of individual Fabs was measured using a VP-DSC system (MicroCal, Northampton, MA). All calorimetric scans were performed in a buffer composed of 10 mM NaH2PO4 (pH 7.5), 150 mM NaCl, and 10% glycerol. Protein samples at 10 μM were heated from 30 to 90°C at a scanning rate of 1°C min−1. The ORIGIN software package (MicroCal) was used for data collection and analysis. The buffer baseline was subtracted from the raw data, which were normalized by protein concentration to obtain thermodynamic parameters and then fitted to a to a non-two-state model.
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