The titration of compounds binding to cMyBP-C was performed on an Affinity ITC (TA Instruments). Each compound in M-ABB buffer (2000–3000 μM) was titrated into C0-C2 in M-ABB buffer (100 μM, 350 μl). For the titration, a preliminary 2.5-μl injection (ignored in data analysis) was followed by 19–39 subsequent 2.5-μl injections with 4 min waits between each injection. The reference cell was filled with distilled water. To account for heat changes of dilution and mechanical mixing, binding data were corrected by subtraction of the final plateau injection values (when binding is complete) prior to curve fitting. Corrected binding data was processed using the NanoAnalyze analysis software (TA Instruments).
Affinity itc
Manufactured by TA Instruments
Sourced in United States
The Affinity ITC (Isothermal Titration Calorimetry) is a laboratory instrument designed to measure the heat effects associated with interactions between molecules. It provides quantitative information about the thermodynamics of biomolecular interactions, including binding affinity, reaction stoichiometry, and enthalpy changes.
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Lab products found in correlation
Nanoanalyze software, by TA Instruments (9 mentions)
Nanodrop one, by Thermo Fisher Scientific (4 mentions)
Nanoanalyze, by TA Instruments (3 mentions)
Nanoanalyze data analysis, by TA Instruments (2 mentions)
Nuclepore track etch membrane, by Cytiva (2 mentions)
Nanoanalyzetm software, by TA Instruments (1 mentions)
Avance 2 600 mhz, by Bruker (1 mentions)
Dfs high resolution gc ms, by Thermo Fisher Scientific (1 mentions)
Nano itc, by TA Instruments (1 mentions)
Origin software, by Malvern Panalytical (1 mentions)
Vp itc, by Malvern Panalytical (1 mentions)
Phytic acid sodium salt hydrate, by Merck Group (1 mentions)
Cytisine, by Merck Group (1 mentions)
Origin 2016, by OriginLab (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
41 protocols using affinity itc
1
Quantifying cMyBP-C Compound Binding
2
Calorimetric Analysis of Protein-Ligand Interactions
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All calorimetric experiments were carried out in 20 mM Hepes (pH 7.5), 150 mM NaCl, 0.5 mM TCEP, and 3.0% DMSO at 25°C using an Affinity ITC from TA Instruments (New Castle, DE) equipped with autosampler. Short protein or buffer (10 μM; for control) in the calorimetric cell was titrated by injecting 3 μl of 100 μM ligand solution in 200-s intervals with stirring speed at 75 rpm. The resulting isotherms were subtracted against buffer runs and fitted with a single-site model to yield thermodynamic parameters of ΔH and ΔS, stoichiometry, and KD using NanoAnalyze software (TA Instruments).
3
Ligand Binding Affinity to NR4A1 by ITC
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Isothermal titration calorimetry (ITC) was used to determine the ligand-binding constant (KD) for binding to NR4A1 utilizing an Affinity ITC (TA Instruments, New Castle, DE, USA). Briefly, the experimental setup was as follows. The ITC sample cell contained 250 µL or NR4A1 protein (ligand-binding domain: LBD) at a concentration of 20 µmol/L in a buffer containing 20 mmol sodium phosphate/L (pH 7.4), 5% glycerol, and 1.0% ethanol. The ligand titrant was prepared in the same budder as that above at a ligand concentration of 100 µmol/L. The initial ligand stock solution was prepared at a final concentration of 10 mmol ligand/L ethanol prior to the preparation of the ligand titrant. Ligand titration into protein was performed at 25 °C with a stir rate of 125 rpm. Each ligand injection volume was 5 µL followed by a duration of 200 s to measure the total heat flow required to maintain constant temperature. A total of thirty injections were carried out for each ligand/NR4A1 combination. In a separate set of injections, the same ligand dilution was placed into the buffer. The ligand/buffer values were subtracted from the ligand/protein values prior to data analysis using the Affinity ITC manufacturer-supplied data analysis software package. The resulting data are plotted as heat flow (µJ) versus the molar ratio of the injected ligand to NR4A1 in the sample cell.
4
Isothermal Titration Calorimetry of HNF4α
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Isothermal titration calorimetry was performed on an Affinity ITC (TA Instruments, New Castle, DE, USA). The instrument was equilibrated to 25 °C and the stirring rate was set to 75 rpm. HNF4α buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM TCEP, 5% w/v glycerol) with up to 1% DMSO (final concentration) was used for ITC. The recombinant HNF4α LBD protein (10–20 µM, 172 µL) was titrated with fragments (4 , 6 , 7 , 9 , 10 ; 50–200 µM; dissolved in the same buffer). A total of 20-30 injections with a volume of 2.5–4 µL and with an interval of 200–300 s were performed. As a control experiment, HNF4α protein was titrated with buffer, and the buffer was titrated with test compound to detect dissolution artifacts with otherwise identical experimental parameters. Data analysis was performed with NanoAnalyzeTM Software (TA Instruments, New Castle, DE, USA.) using an independent binding model.
5
Pillar[5]arene Synthesis and Characterization
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Nuclear magnetic resonance (NMR)
spectroscopy was done using a Bruker Avance II 600 MHz (Germany) spectrometer.
Electron impact ionization (EI) mass spectrometry was performed using
a Thermo Scientific DFS high-resolution GC/MS (Germany) mass spectrometer.
Electrospray ionization in high-resolution mode was done using a Waters
Xevo G2-S QToF, (Germany) LC MS/MS mass spectrometer. The single crystal
data collections were made on Bruker X8 Prospector (Germany) and Rigaku
Rapid II (Japan) diffractometers. ITC studies were carried out on
an Affinity ITC, TA Instruments. DLS analysis was done on a Zetasizer
Nano Range, Malvern PANalytical system (U.K.). Flash column chromatography
was performed using silica gel (Silica gel 60, 40–60 mesh ASTM,
EMD Millipore, Merck KGaA, Germany). DMF is distilled before use.
All other reagents and solvents were of reagent grade purity and used
without further purification. The synthesis of 1,4-bis(2-bromoethoxy)benzene,
1-(1,4-bis(2-bromoethoxy))-2,3,4,5-dimethoxy-pillar[5]arene,1,1-Pillar-a , 1,2-(1,4-bis(2-bromoethoxy))-3,4,5-dimethoxy-pillar[5]arene, 1,2-Pillar-a , and 1,3-(1,4-bis(2-bromoethoxy))-2,4,5-dimethoxy-pillar[5]arene, 1,3-Pillar-a have been previously discussed.31 (link)
spectroscopy was done using a Bruker Avance II 600 MHz (Germany) spectrometer.
Electron impact ionization (EI) mass spectrometry was performed using
a Thermo Scientific DFS high-resolution GC/MS (Germany) mass spectrometer.
Electrospray ionization in high-resolution mode was done using a Waters
Xevo G2-S QToF, (Germany) LC MS/MS mass spectrometer. The single crystal
data collections were made on Bruker X8 Prospector (Germany) and Rigaku
Rapid II (Japan) diffractometers. ITC studies were carried out on
an Affinity ITC, TA Instruments. DLS analysis was done on a Zetasizer
Nano Range, Malvern PANalytical system (U.K.). Flash column chromatography
was performed using silica gel (Silica gel 60, 40–60 mesh ASTM,
EMD Millipore, Merck KGaA, Germany). DMF is distilled before use.
All other reagents and solvents were of reagent grade purity and used
without further purification. The synthesis of 1,4-bis(2-bromoethoxy)benzene,
1-(1,4-bis(2-bromoethoxy))-2,3,4,5-dimethoxy-pillar[5]arene,
6
Affinity Kinetics of BK1.3 and CCL8 Interaction
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The experiments were carried out on a TA Instruments Affinity ITC with low volume cells at 25 °C and stirring at 250 rpm using a buffer system of 50 mm HEPES, 150 mm NaCl, pH 7.5. Twenty sequential 2-μl injections of BK1.3 (125 μm ) into CCL8 (10 μm ) were used to generate a binding isotherm for the interaction, which was fitted to the independent (single-site) model using NanoAnalyze (version 3.11.0).
7
Thermodynamic Characterization of Protein-DNA Binding
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Isothermal titration calorimetry (ITC) experiments were performed at 20°C in a buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl and 0.5 mM TCEP using the Affinity ITC instrument (TA Instruments, New Castle, DE). The proteins at 160–240 µM were titrated into the reaction cell containing ssDNA at 40–50 µM using the parameters of 3 µl injection with a total of 30 serial injections. The integrated heat of binding after correction was fitted using an independent single binding site model, based on the manufacturer's protocol, from which thermodynamic binding parameters (ΔH and TΔS), equilibrium association and dissociation constants (KA and KD) and stoichiometry (n) were calculated.
8
Isothermal Titration Calorimetry of Vancomycin-Aptamer Interactions
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We used an Affinity ITC (TA Instruments, New Castle, DE) coupled to an autosampler and associated software to run all ITC titrations. We used PBS + 2 mM MgCl 2 as a supporting electrolyte to prepare vancomycin and aptamer solutions, and removed air bubbles by keeping the solutions under vacuum for 10 min prior to the ITC experiments. Before each titration, we purged the system three times with deionized water, and let it equilibrate in buffer for 300 s before the first injection. We loaded the syringe with a 300 μM vancomycin solution and the sample cell with at 20 μM solution of the different sequences tested. We recorded the heat-exchanged after the injection of 1 μL of the vancomycin solution for a total of 30 times, allowing the system to equilibrate between 150 and 180 s after each injection. All titrations were done at 25 °C and performed in triplicate.
9
Isothermal Titration Calorimetry of EF-Tu and EF-Ts
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ITC titrations were carried out on an Affinity ITC (TA Instruments). Before the measurement, nucleotide-free EF-Tu, pEF-TuT382, and EF-Ts were dialyzed to 50 mM Hepes (pH 7.4), 40 mM NH4Cl, 2 mM MgCl2, and 1 mM TCEP. In each case, nucleotides were prepared with buffer from the last step of protein dialysis. The samples were filtered and degassed for 10 min before being examined in the calorimeter, and the titrations were performed at temperatures ranging from 10° to 35°C. All the experiments consisted of injection of constant volumes of 2 μl of titrant into the cell (200 μl) with a stirring rate of 75 rpm. Nominal sample concentrations were between 5 and 20 μM in the cell and 100 and 500 μM in the syringe. Actual sample concentrations were determined after dialysis or buffer exchange by measurement of their absorption at 280 nm. All data were analyzed using the MicroCal Origin ITC 7.0 and NanoAnalyze software packages. Binding affinities are reported in table S1.
10
SFPQ Peptide Binding Calorimetry
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All calorimetric experiments were performed in 20 mM Hepes (pH 7.5), 150 mM NaCl, and 0.5 mM TCEP, with 2% DMSO at 25°C using an Affinity ITC from TA Instruments equipped with autosampler. Briefly, 350 µl of buffer or protein at 20 µM was placed into the calorimetric cell, and 250 µl of various SFPQ peptides (KE BioChem) at 200 µM were loaded into titration syringe. 4 µl syringe solution was injected into the calorimetric cell 30 times with a 200-s interval between injections. Thermodynamic parameters (Kd, stoichiometry, and enthalpy) were calculated according to the single-site model provided in NanoAnalyze software (TA Instruments).
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