nano itc instrument, by TA Instruments - Product details

1

TgPDCD5 Heparin Binding Kinetics

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Quantitative estimations of heparin-binding
affinities for some proteins using ITC have been reported.^{49 (link)−51 (link)} To investigate the interaction between TgPDCD5 and heparin sulfate
(H3393, Sigma-Aldrich) or Enoxaparin (E0180000, Sigma-Aldrich), ITC
was performed using a Nano ITC instrument (TA Instruments). Aliquots
of 4 μL of 3 mM TgPDCD5 were injected into 0.12 mM heparin sulfate
or Enoxaparin in 25 mM phosphate buffer at pH 4.5 with 100 mM NaCl
while maintaining a temperature of 37 °C with 250 rpm stirring.
Background heat from the protein to buffer titrations was subtracted.
The thermal parameters (enthalpy ΔH and entropy
ΔS), stoichiometry of the binding (n), and dissociation constant (K_d) were derived by fitting the data to an independent binding model
using Launch NanoAnalyze v2.3.6 software.

Lin G.M., Yu T.A., Chang C.F, & Hsu C.H. (2024). Proline Isomerization and Molten Globular Property of TgPDCD5 Secreted from Toxoplasma gondii Confers Its Regulation of Heparin Sulfate Binding. JACS Au, 4(5), 1763-1774.

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2

Protein-Peptide Binding Affinity Assay

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The experiments were conducted using a Nano ITC instrument (TA instruments, Utah USA). Purified proteins were dialyzed against ITC buffer (20 mM sodium phosphate pH 7.5 and 100 mM NaCl). For these experiments, peptides used were used at a concentration of 200 μM in the calorimetric cell and titrated with 2 mM of various protein at 20 °C. Typically for each experiment, 25 injections (2 μL of protein sample) at 200 s intervals were performed. Binding isotherms were analyzed according to 1:1 binding model using NanoAnalyze software.

Klein G., Wojtkiewicz P., Biernacka D., Stupak A., Gorzelak P, & Raina S. (2020). Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli. International Journal of Molecular Sciences, 21(12), 4212.

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3

Chloride Binding to CLC-ec1 Mutant

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Cl^– binding to the A399C/A432C mutant of CLC–ec1 was carried out as described^{36 (link),38 (link)} using a nanoITC instrument (TA Instruments). For these experiments, the final purification step of the protein was purified over a gel filtration column pre-equilibrated in 100 mM Na-K-Tartrate, 20 mM Hepes, 50 μM DMNG, pH 7.5 (Buffer B₀) and concentrated to 50-195 μM. The A399C/A432C mutant was not stable in the absence of Cl^– upon incubation with Hg²⁺. Therefore, in this case the Hg²⁺ reaction was carried out in Buffer B. Excess Cl^– and Hg²⁺ were successively removed by running the protein over a gel filtration column pre-equilibrated in buffer B₀. The injection syringe was filled with buffer B₀ with 50 mM KCl added, to achieve final Molar Ratios of 100-250. Each experiment consisted of 30-48 injections of 1 μl of the ligand solution at 3-4 min intervals into the experimental chamber kept under constant stirring at 350 rpm and at 25.0±0.1 °C. All solutions were filtered and degassed prior to use. The ITC data was fit to a single site Wiseman isotherm as described^{36 (link),38 (link)} using the NanoAnalyze program from TA instruments.

Basilio D., Noack K., Picollo A, & Accardi A. (2014). Conformational changes required for H+/Cl– exchange mediated by a CLC transporter. Nature structural & molecular biology, 21(5), 456-463.

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4

Calorimetric Analysis of Dileucine Peptide Binding

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ITC experiments were conducted on a NanoITC instrument (TA
Instruments) at 20°C. A model dileucine peptide (DSVIL) was
dissolved in 20 mM sodium phosphate (pH 6.5), 200 mM NaCl, 0.5 mM TCEP. A
PD-10 desalting column (GE Healthcare) was used to buffer exchange human
tVHS (residues 220-360) into the same buffer as the peptide. Incremental
titration was performed with an initial baseline of 180 s and injection
intervals of 180 s. Peptide was titrated in to a ratio of 6x molar excess.
Data were analyzed in NanoAnalyze (TA Instruments).

Archuleta T.L., Frazier M.N., Monken A.E., Kendall A.K., Harp J., McCoy A.J., Creanza N, & Jackson L.P. (2017). Structure and evolution of ENTH and VHS/ENTH-like domains in tepsin. Traffic (Copenhagen, Denmark), 18(9), 590-603.

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5

Chloride Binding to CLC-ec1 Mutant

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Cl^– binding to the A399C/A432C mutant of CLC–ec1 was carried out as described^{36 (link),38 (link)} using a nanoITC instrument (TA Instruments). For these experiments, the final purification step of the protein was purified over a gel filtration column pre-equilibrated in 100 mM Na-K-Tartrate, 20 mM Hepes, 50 μM DMNG, pH 7.5 (Buffer B₀) and concentrated to 50-195 μM. The A399C/A432C mutant was not stable in the absence of Cl^– upon incubation with Hg²⁺. Therefore, in this case the Hg²⁺ reaction was carried out in Buffer B. Excess Cl^– and Hg²⁺ were successively removed by running the protein over a gel filtration column pre-equilibrated in buffer B₀. The injection syringe was filled with buffer B₀ with 50 mM KCl added, to achieve final Molar Ratios of 100-250. Each experiment consisted of 30-48 injections of 1 μl of the ligand solution at 3-4 min intervals into the experimental chamber kept under constant stirring at 350 rpm and at 25.0±0.1 °C. All solutions were filtered and degassed prior to use. The ITC data was fit to a single site Wiseman isotherm as described^{36 (link),38 (link)} using the NanoAnalyze program from TA instruments.

Basilio D., Noack K., Picollo A, & Accardi A. (2014). Conformational changes required for H+/Cl– exchange mediated by a CLC transporter. Nature structural & molecular biology, 21(5), 456-463.

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6

Thermodynamic Analysis of Rubisco Binding

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The same, commercially obtained spinach Rubisco (Sigma-Aldrich; catalogue # R8000; S1 Fig; specific activity 0.05 units / mg) was inactivated by dissolving in 10 mM HEPES buffer pH 7.9 containing 100 μM EDTA and dialyzing twice against 2 L of ITC buffer (10 mM HEPES buffer pH 7.9) using a 3,500 MWCO dialysis membrane. A low volume Nano ITC instrument (TA Instruments) with a cell volume of 190 μL was used for this study. Stock solutions of RuBP (100 mM) and (+)-ABA (10 mM) were diluted to the working concentration using ITC buffer. The ITC experiments were performed at 20°C. Rubisco, ligands and buffer were equilibrated at 20°C and degassed before the experiment. A 50 μL syringe was used to deliver 19 injections (2.5 μL each) of the ligands (0.75 mM RuBP or 5 mM (+)-ABA) into the cell containing either ITC buffer or Rubisco (39.42 μM). The first injection of 0.71 μL was excluded from data processing. A stir rate of 200 rpm was used to mix the reactants continuously. The data were processed using the NanoAnalyze software (TA Instruments) using the ‘Independent binding’ model.

Galka M.M., Rajagopalan N., Buhrow L.M., Nelson K.M., Switala J., Cutler A.J., Palmer D.R., Loewen P.C., Abrams S.R, & Loewen M.C. (2015). Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase. PLoS ONE, 10(7), e0133033.

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7

Fur Protein Binding Kinetics

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Experiments were performed on a Nano ITC instrument (TA Instruments) at 20 °C. WT or mutant Fur proteins were diluted (60 μM) and titrated against a buffer of Tris-HCl (20 mM, pH 8.0), NaCl (500 mM) and MnCl₂ (2–10 mM). ITC data were processed with NanoAnalyze software.

Deng Z., Wang Q., Liu Z., Zhang M., Machado A.C., Chiu T.P., Feng C., Zhang Q., Yu L., Qi L., Zheng J., Wang X., Huo X., Qi X., Li X., Wu W., Rohs R., Li Y, & Chen Z. (2015). Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator. Nature Communications, 6, 7642.

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8

Quantifying Fc gamma RIIIA-Trastuzumab Binding

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Affinity constants under equilibrium (K_a) were obtained from a Nano ITC instrument (TA Instruments Inc.; New Castle, DE). 300 μL of FcγRIIIa solutions at 5.0 μM in PBS at pH 7.2 was titrated with continuous injections of 1.9 μL trastuzumab solutions at 50 μM in PBS at pH 7.2 until saturation at 25°C. NanoAnalyze Software v2.4.1 (TA Instruments Inc.; New Castle, DE) was used for the integration of heat signals and nonlinear regression analysis of the data.

López-Morales C.A., Miranda-Hernández M.P., Juárez-Bayardo L.C., Ramírez-Ibáñez N.D., Romero-Díaz A.J., Piña-Lara N., Campos-García V.R., Pérez N.O., Flores-Ortiz L.F, & Medina-Rivero E. (2015). Physicochemical and Biological Characterization of a Biosimilar Trastuzumab. BioMed Research International, 2015, 427235.

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9

Isothermal Titration Calorimetry of FOX-DBD Binding

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ITC analyses were performed at 25°C using NANO ITC instrument (TA Instruments). The purified protein was placed in ITC buffer (20 mM HEPES, pH7.5, 250 mM NaCl, 10 mM MgCl₂ and 1 mM TCEP) at the concentrations of 25–40 μM. DNA duplex was dissolved in the same buffer to the concentrations of 90–180 μM. The DNA was titrated in duplicate into FOX-DBDs using 2 μl injections with 300 s intervals. Data were processed using the launch NanoAnalyze software.

Li J., Dai S., Chen X., Liang X., Qu L., Jiang L., Guo M., Zhou Z., Wei H., Zhang H., Chen Z., Chen L, & Chen Y. (2021). Mechanism of forkhead transcription factors binding to a novel palindromic DNA site. Nucleic Acids Research, 49(6), 3573-3583.

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10

Thermodynamic Characterization of Compound 10

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ITC measurements were carried out at 25°C using a Nano-ITC instrument (TA instrument) with stirring at 300 rpm. Tagged protein sample in 25 mM Tris buffer pH 7.5, 75 mM NaCl, 5 mM MgCl₂ and 1 mM DTT was dialyzed overnight into 1 L of 20 mM Hepes 7.5, 50 mM NaCl, 1 mM MgCl₂ and 0.8 % DMSO. Protein and ligand concentration (compound 10) were adjusted to 90 μM and 800 μM, respectively with the dialysis buffer and degassed for 5 minutes prior to usage. Aliquot (2.5 μL) of the ligand was injected into the protein sample until binding appeared saturated (20 injections, at 300 s intervals). Titrations of ligand buffer-only sample was performed to provide baseline readings. The corrected heat change was fitted with an independent binding model in the NanoAnalyze software (TA instrument) to obtain binding affinity constants (K_d), enthalpy and entropy of binding (ΔH and ΔS) as well as the stoichiometry (n) of binding

Krucinska J., Falcone E., Erlandsen H., Hazeen A., Lombardo M.N., Estrada A., Robinson V., Anderson A.C, & Wright D.L. (2019). Structural and Functional Studies of Bacterial Enolase, a Potential Target Against Gram-Negative Pathogens.. Biochemistry, 58(9), 1188-1197.

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Nano itc instrument

Lab products found in correlation

41 protocols using nano itc instrument

TgPDCD5 Heparin Binding Kinetics

Protein-Peptide Binding Affinity Assay

Chloride Binding to CLC-ec1 Mutant

Calorimetric Analysis of Dileucine Peptide Binding

Chloride Binding to CLC-ec1 Mutant

Thermodynamic Analysis of Rubisco Binding

Fur Protein Binding Kinetics

Quantifying Fc gamma RIIIA-Trastuzumab Binding

Isothermal Titration Calorimetry of FOX-DBD Binding

Thermodynamic Characterization of Compound 10

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