ITC measurements with a Nano Isothermal Titration Calorimeter ( TA Instruments) and data process using the NanoAnalyze version 2.3.6 software29 (link), 30 were performed as described.16 (link), 28 (link) MelB in 20 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, 10% glycerol, and a given detergent was placed into the sample cell, and melibiose and IIAGlc were prepared in the same buffer used for the permease. Data fitting using one-site independent binding model31 yields the association constant (Ka) and enthalpy change ΔH values. ΔG = - RT ln Ka. The dissociation constant (Kd) = 1/Ka. Entropy change (−TΔS) is calculated from equation ΔG = ΔH - TΔS.
Nano isothermal titration calorimeter
Manufactured by TA Instruments
Sourced in United States
The Nano Isothermal Titration Calorimeter is a laboratory instrument designed to measure the heat effects associated with interactions between a titrant and a sample. It provides high-sensitivity measurements of the thermodynamic properties of biomolecular interactions, chemical reactions, and other physical processes.
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14 protocols using nano isothermal titration calorimeter
1
Isothermal Titration Calorimetry of Melibiose-Permease Interactions
2
Measurement of MERS-CoV Macro Domain Binding to ADP-Ribose
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Binding of ADP-ribose to the MERS-CoV macro domain was measured by ITC
with the Nano Isothermal Titration Calorimeter (TA Instruments).
Aliquots of 3 μl of 1.14 mm ADP-ribose were titrated by
injection into protein (0.057 mm in 0.98 ml) in 20 mm Tris-HCl (pH 7.0) and 100 mm NaCl. Experiments were carried out
at 25 °C with 250 rpm stirring. Background heat from ligand to
buffer titrations was subtracted, and the corrected heat from the
binding reaction was used to derive values for the stoichiometry of the
binding (n), Kd, apparent
enthalpy of binding (ΔH), and entropy change
(ΔS). Data were fitted by use of an
independent binding model with Launch NanoAnalyze version 2.3.6.
with the Nano Isothermal Titration Calorimeter (TA Instruments).
Aliquots of 3 μl of 1.14 m
injection into protein (0.057 m
at 25 °C with 250 rpm stirring. Background heat from ligand to
buffer titrations was subtracted, and the corrected heat from the
binding reaction was used to derive values for the stoichiometry of the
binding (n), Kd, apparent
enthalpy of binding (ΔH), and entropy change
(ΔS). Data were fitted by use of an
independent binding model with Launch NanoAnalyze version 2.3.6.
3
Thermodynamics of PARG Binding to ADP-ribose
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Binding of ADP-ribose to wild-type or mutant DrPARG was measured by ITC with the Nano Isothermal Titration Calorimeter (TA Instruments). Aliquots of 4 μL of 1.5–3 mM ADP-ribose were injected into sample cells containing 0.075–0.15 mM protein sample in 20 mM Tris-HCl, pH 7.0, and 100 mM NaCl. ITC experiments were executed at 25 °C with 250-r.p.m. stirring speed. Additional background heat from titration of ADP-ribose to buffer was subtracted during data analysis. The corrected heat was used for deriving parameters of stoichiometry of the binding (n), association constant (Ka), dissociation constant (Kd), apparent enthalpy of binding (ΔH), and entropy change (ΔS). Data were fitted by applying an independent binding model with NanoAnalyze v2.3.6.
4
Calorimetric Characterization of Melibiose Binding to MelB
5
Characterization of eIF4A Binding
6
Nano-ITC Calorimetric Experiments in Phosphate Buffer
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The nanoITC experiments were performed at 298 K with a nano-isothermal titration calorimeter (TA Instruments, New Castle, USA). All samples were degassed (degassing time t = 15 min) using Degassing Station (TA Instruments, New Castle, USA). Calorimetric measurements were determined based on the experimental and theoretical parameters [15 (link), 16 (link), 25 (link)–28 (link)] (Table 1 ). All solutions were prepared using a phosphate buffer (0.05 M, pH 7.4).
2 ) [29 (link)] where ΔH is enthalpy change [kcal × mol−1]; T is temperature [K]; ΔS is entropy change [kcal × mol−1 K−1].
Experimental dataset
| Property | Value |
|---|---|
| Syringe concentration (10–3 mol/dm3) | 1.2 |
| Cell concentration (10–5 mol/dm3) | 3 |
| Initial cell Vol (μl) | 300 |
| Inj. interval (s) | 180 |
| Inj. volume (μl) | 2.38 |
| Temperature (K) | 298 |
| Stir rate (RPM) | 300 |
7
Calorimetric Titration of NanR with Neu5Ac
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Calorimetric titrations of NanR with Neu5Ac were performed with a Nano Isothermal Titration Calorimeter (TA Instruments). Purified NanR was initially concentrated to a final concentration of 416 µM via centrifugal ultrafiltration (30 kDa molecular weight cutoff; Sartorius) and then extensively dialyzed against buffer C. Neu5Ac was prepared in the same buffer by diluting a 100 mM stock solution to a final concentration of 1 mM. Protein sample (200 µL) was loaded in the sample cell, and 50 µL of Neu5Ac was loaded into the injection syringe. Titrations were initiated by a 1 µL injection, followed by 24 consecutive 2 μL injections every 200 s at 8 °C and a constant stirring speed of 60 rpm. A blank correction was obtained by injection of Neu5Ac (1 mM) into buffer C using an identical set-up. Titration data were integrated using NITPIC59 (link),60 (link) and analyzed in SEDPHAT by discarding the initial injection and fitting the binding isotherm 1:1 interaction model61 (link) to obtain KD values.
8
Melibiose Binding Kinetics of MelB_St
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ITC measurements were performed in a nano isothermal titration calorimeter (TA Instruments). The purified MelBSt in the dialysis buffer was injected into the ITC sample cell and 6 mM melibiose dissolved in the same dialysis buffer was titrated incrementally into the protein sample, and the heat rate was recorded at 25 °C. Melibiose binding was also measured in the presence of 100 mM LiCl. The cumulative heat change (ΔQ) was plotted against the molar ratio of melibiose to MelBSt and fitted with the one-site independent binding model (NanoAnalyse software).
9
Binding Kinetics of PERK-LD to CNPY2 using ITC
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ITC experiments were performed using a nano Isothermal Titration Calorimeter from TA instruments (Fig. 3d , 4b and Supplementary Fig. 4 ). Solutions were thoroughly degassed prior to experiments to avoid air bubbles in the calorimeter. All protein samples were prepared in Phosphate Buffered Saline. For binding of PERK luminal domain (PERK-LD) to PBS buffer, CNPY2 or CNPY2 mutant protein, typically 50 μM of CNPY2 or CNPY2 mutant protein was placed in the reaction cell in a 300 μl volume, and 500 μM PERK-LD was placed in the ITC syringe. Aliquots of 1.5 μl were injected into the reaction cell at 300 seconds (s) intervals with a stirring speed of 300 round per minute and temperature of 20°C. The titrations were completed after 32 injections. The data was then analyzed using the software Nano Analyzer v2.1.13.
10
Harmine Binding to DNMT3B-3L by ITC
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Binding of harmine
to DNMT3B-3L was measured by ITC with the Nano Isothermal Titration
Calorimeter (TA Instruments). Aliquots of 7 μL of 0.52 mM harmine
were titrated by injection into protein (0.02 mM in 1.03 mL cell)
in a buffer containing 20 mM Tris-HCl, at pH 8.0, 100 mM NaCl, and
0.2 mM TCEP. Background heat from ligand to buffer titrations was
subtracted, and the corrected heat from the binding reaction was used
to derive values for the stoichiometry (n), dissociation
constant (Kd), apparent enthalpy of binding
(ΔH), and entropy change (ΔS). All experiments were performed in triplicate. Data were fitted
by an independent binding model with NanoAnalyze version 3.12.5.
to DNMT3B-3L was measured by ITC with the Nano Isothermal Titration
Calorimeter (TA Instruments). Aliquots of 7 μL of 0.52 mM harmine
were titrated by injection into protein (0.02 mM in 1.03 mL cell)
in a buffer containing 20 mM Tris-HCl, at pH 8.0, 100 mM NaCl, and
0.2 mM TCEP. Background heat from ligand to buffer titrations was
subtracted, and the corrected heat from the binding reaction was used
to derive values for the stoichiometry (n), dissociation
constant (Kd), apparent enthalpy of binding
(ΔH), and entropy change (ΔS). All experiments were performed in triplicate. Data were fitted
by an independent binding model with NanoAnalyze version 3.12.5.
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