Microcal peaq itc analysis
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The MicroCal PEAQ-ITC analysis system is a laboratory instrument designed for isothermal titration calorimetry (ITC) measurements. It is used to characterize the thermodynamics of biomolecular interactions, including binding affinities, stoichiometry, and enthalpy changes.
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15 protocols using microcal peaq itc analysis
1
Biophysical Characterization of BabA-IgG Binding
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ITC was performed on an Auto-ITC200 from Malvern Panalytical (Malvern, UK). Recombinant BabA from H. pylori 17875/Leb was stabilized by adding hexa-lysine and myc-tag to the BabA protein C-terminus as described previously 25 (link). Experiments were performed at 25°C in PBS with 10 µM of BabA and injection of 50 µM ABbA-IgG. The titrations were repeated three times with high feedback and a filter period of 5 s. For each experiment, 19 automated injections of 2 μL each were performed (duration 0.8 s) with 300 s intervals between each injection with a stirring speed of 1000 rpm. Calorimetric data were plotted and fitted using a single-site binding model with MicroCal PEAQ-ITC Analysis from Malvern Panalytical (Malvern, UK).
SPR experiments were performed on a Biacore 3000 system (GE Healthcare, Uppsala, Sweden). ABbA-IgG was immobilized on a CM5 chip at around 50 RU levels by standard amine coupling at pH 5. Recombinant BabA from H. pylori 17875/Leb was injected for 2 min over the surface at concentrations of 66 nM or 40 nM at two- or three-fold dilutions in PBS + 0.005% Tween-20 at 25°C. Regeneration was done by injection of 10 mM glycine (pH 1.7) for 18 s. Dissociation and rate constants were determined by global fitting with Scrubber2 software (Biological Software, Australia). The experiment was repeated three times and presented as the average value.
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2
Thermodynamic Analysis of ApoE Isoform Interactions
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ApoE isoforms, as well as truncated ApoE4 (ApoE41-191 were dialyzed against PBS + 0.01% (v/v) Triton X-100 overnight at 4 °C using 3.5 K Slide-A-LyzerTM dialysis cassettes (ThermoFisher Scientific, Loughborough, UK). ApoE, as well as ApoE41-191 at 300 μM was injected into the cell containing compound at 25–50 μM in PBS + 0.01% (v/v) Triton X-100 + 2% (v/v) DMSO. Titration experiments were carried out on a Microcal PEAQ-ITC (Malvern Panalytical, Malvern, UK) at 25 °C and 750 rpm, and consisted of an initial (1 μL) injection followed by additional 18 (2 μL) injections. Injection of the protein into PBS + 0.01% (v/v) Triton X-100 + 2% (v/v) DMSO was used as control experiment and integrated control heats were subtracted from the reaction heats. Data were analyzed on Microcal PEAQ-ITC analysis software (Malvern, version 1.1.0.1262) and data-fitted for one set of sites. Binding affinities as presented in Table 1 are the average of two independent experiments (±SD).
3
Thermodynamic analysis of FOXO1-ETS1-DNA complexes
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Titrations were carried out using either a MicroCal PEAQ-ITC or a MicroCal iTC200 calorimeter at 25 °C. All protein and DNA samples were dialyzed overnight at 4 °C against the same buffer containing 50 mM Bis–Tris, pH 6.5, and 150 mM NaCl. Concentrations used in each experiment are listed in Supplementary Data 3 . For the FOXO1 titrations against the DNAs with or without ETS1 20 injections of titrant were made at 120 s intervals, while stirring at 750 rpm. The first injection was 0.4 µl, and 2 µl for the remaining nineteen. For the ETS1 titrations against the different DNAs 13 injections of 3 µl were done. While ETS1 was kept in the cell and DNA was added from the syringe, FOXO1 was always titrated from the syringe into the cell filled with DNA or DNA and ETS1 in equimolar ratios. Data were reduced with heat spikes from control and baseline corrected. The raw data integration, normalization and titration curve fitting was done using the MicroCal PEAQ-ITC analysis software provided by Malvern.
4
Isothermal Titration Calorimetry of Chitohexaose and N-plug Binding to VhChiP
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ITC experiments were carried out at least three times using the MicroCal PEAQ-ITC (Malvern Instruments Ltd) at 25 ± 1 °C with a stirring speed of 500 rpm. For titration experiments, 40 μl of chitohexaose or the N-plug was titrated from a syringe into the 300 μl-calorimeter cells containing 0.05% (v/v) LDAO, 20 mM sodium phosphate buffer, pH 7.5, and protein solution. The optimized concentration ratios of chitohexaose and N-plug to VhChiP variants are shown in Table 3 . Injections (2 μl per injection) were repeated 19 times over 150-s intervals. The background was measured by injecting the corresponding ligand into the cell containing only the buffer. The ITC data were collected and analyzed using the MicroCal PEAQ-ITC analysis software (https://www.malvernpanalytical.com/en/support/product-support/software/microcal-peaq-itc-analysis-software-v141 ). The ITC profile obtained by injecting the corresponding ligand into the reaction cell containing buffer without VhChiP was subtracted from the control dataset. The resultant data were fitted by a single-site binding model with the nonlinear least square algorithm.
5
Quantifying HMGB1-F1,6P Interactions
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ITC experiments were carried out using a Malvern‐PEAQ apparatus. Typical titration experiment consisted of 19 or 13 consecutive injections at 150 s intervals into the titration cell. The F1,6P, peptide, and protein (HMGB1∖HMGB1‐A box) were dissolved in the same buffer (100 mm Hepes, 50 mm NaCl) and degassed for 10 min under vacuum in each experiment. Controls included F1,6P injected into buffer, peptide injected into buffer and F1,6P‐peptide injected into buffer solutions, respectively. Data were analyzed by using nonlinear regression with a single site binding model in Malvern MicroCal PEAQ ITC Analysis.
6
Thermodynamic Analysis of Protein-Ligand Interactions
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Experiments were performed using a Microcal PEAQ-ITC (Malvern Instrument GmbH). The protein was dialyzed overnight at 4°C against the ITC buffer (20 mM HEPES at pH 7.5, 150 mM NaCl, 5 mM TCEP). The Tm1 WT protein in the syringe, at a concentration of 1 mM, was injected into the cell containing the buffer. Titrations were performed at 20°C or 25°C and consisted of 13 or 25 injections. The data were fitted using the dissociation fitting model of the Microcal PEAQ-ITC analysis software (Malvern).
7
Determining Binding Constants via ITC
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Binding constants were determined by means of ITC measurements with a MicroCal PEAQ‐ITC system (Malvern). Solutions of protein (50–100 μm ) and 4 e (1.0 mm ) were prepared by using the same buffer stock (≈300 mm Tris⋅ HCl, pH 8.0). Three control experiments, namely the titration of buffer with buffer, the titration of protein solution with buffer, and the titration of buffer with ligand, were performed. Each titration experiment was baseline corrected and the signal peaks of each injection were integrated by MicroCal PEAQ‐ITC analysis (version 1.22, Malvern Panalytical). Then, each protein–ligand titration experiment was corrected for each control experiment and processed by using the built‐in functions of MicroCal PEAQ‐ITC analysis with the “single set of identical sites” option. Thus, a ΔH versus molar ratio (ligand:protein) plot and fit values for the binding constant KD, the number of binding sites n, and the molar heat of ligand binding ΔH were obtained. The fitted number of binding sites in initial analyses was close to one. For better comparability, the number of binding sites was then set to one for all experiments.
8
Thermodynamic Characterization of P450cam
9
Calorimetric Analysis of MAO-A Interactions
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A MicroCal PEAQ-ITC200 calorimeter (Malvern, Worcestershire, UK) was used to study the interactions of MAO-A with the compounds. The enzyme and compounds were prepared in 1% DMSO with methanol. The measurements were performed at 36.6 °C. The stirring speed was set to 307 rpm, and a total of 19 injections were performed within 50 min, with the reference power set to 10.00 μcal/s. All sample solutions were thoroughly degassed before use. The calorimetric cell was filled with a 20 μM (350 µL) MAO-A sample, and the syringe (2 µL of injection volume) was loaded with a 1 mM/L solution or/and 5-HT (1 mM/L). The ITC data were processed using MicroCal PEAQ-ITC analysis software with calorimetric routines. The following parameters were determined: the standard interaction Gibbs energy (ΔG), standard interaction enthalpy (ΔH), entropy (ΔS), equilibrium constant (KD), and stoichiometry of reaction (N) [59 (link)].
10
Thermodynamic Analysis of LgnA-LgnD Binding
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Thermodynamic data of interaction measurements between LgnA and LgnD or truncated LgnDs were generated using a Malvern MicroCal PEAQ-ITC instrument controlled by MicroCal PEAQ-ITC Control Software. Calorimetric titrations were performed with thirteen 3 µL injections with injection duration of 6.0 s, a spacing of 150 s, a reference power of 5 µcal/s, and constant temperature at 25 °C. The concentrations used for titration experiment were LgnA (125 µM) and LgnD (25 µM) or truncated proteins, LgnD-C1-T1 or LgnD-C1-T1-C2 or LgnD-C1 or LgnD-T1, and LgnB (250 µM) with LgnD or truncated LgnD (25 µM). Every set of measurements was repeated in duplicate. Data were analyzed with the “one set binding model” of the software (MicroCal PEAQ-ITC Analysis) provided by the manufacturer. The software calculates the calorimetric binding enthalpy, adsorption constant, number of adsorption sites, and changes in entropy. Enthalpy of dilution effects for solute was considered during ITC measurements.
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