Isothermal titration calorimetry was performed using TA instruments Nano-ITC machine at Yale core facility. 200 µM EPA was injected into 20 µM purified proteins. Data was analyzed using NanoAnalyze software from TA instruments.
Nano itc machine
Manufactured by TA Instruments
The Nano ITC is a high-sensitivity microcalorimeter designed to measure the heat effects associated with molecular interactions. It is capable of determining the thermodynamic parameters of a wide range of chemical and biological processes, including protein-ligand, protein-protein, and protein-DNA interactions.
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7 protocols using nano itc machine
1
Thermodynamic analysis of EPA-protein interactions
2
Thermodynamic Analysis of MxB-Capsid Binding
3
Isothermal Titration Calorimetry of Protein-Peptide Interactions
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Experiments were performed using a Nano ITC machine from TA Instruments. For ITC, proteins were gel filtered into 100 mM tris (pH 7.4), 150 mM NaCl, and 0.25 mM TCEP. Peptides were dissolved in the same buffer. PHear domains at concentrations between 0.10 and 0.15 M were placed in the cell at 12°C, and peptides at concentrations between 1 and 5 mM (depending on a peptide) were titrated with 20 injections of 2.43 μl each separated by 2.5 min. A relevant peptide-into-buffer blank was subtracted from all data, and for constructs, which displayed measurable binding, a minimum of three independent runs that showed clear saturation of binding were used to calculate the mean KD of the reaction, its stoichiometry (n), and their corresponding SEM values. Analysis of results and final figures was carried out using the NanoAnalyzeTM software.
4
Isothermal Titration Calorimetry of PHear Domains
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Experiments were performed using a Nano ITC machine from TA Instruments. For ITC with peptides, PHear domains were gel filtered into 75 mM HEPES pH 7.4, 0.25 mM TCEP. Peptides were dissolved in the same buffer. PHear domains at concentrations between 0.10 and 0.15 M were placed in the cell at 12°C and peptides at concentrations between 1 and 5 mM (depending on a peptide) were titrated in with 20 injections of 2.43 μl each separated by 2.5 minutes. For ITC with WT PHear and Pcore, the proteins were gel filtered into 100 mM TRIS pH 8, 250 mM NaCl, 0.25 mM TCEP and experiments were carried out at 10°C with between 0.05 and 0.1 mM Pcore in the cell and with PHear domain between 2 and 3 mM being titrated in with 20 injections of 2.43 μl each separated by 2.5 minutes. A relevant syringe-solution-into-buffer blank was subtracted from all data and for constructs, which displayed measurable binding, a minimum of three independent runs that showed clear saturation of binding were used to calculate the mean KD of the reaction, its stoichiometry (n), and their corresponding SEM values (see Table S5 ). Analysis of results and final figures were carried out using the NanoAnalyzeTM Software.
5
Biophysical Characterization of TBC1D23 Binding
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Experiments were performed using a Nano ITC machine from TA Instruments. TBC1D23 C-terminal domains were gel-filtered into buffer composed of 100 mM NaCl, 100 mM tris (pH 7.4), and 1 mM tris(2-carboxyethyl)phosphine (TCEP). Peptides were dissolved in the same batch of buffer. Both the wild-type and mutant C-terminal domains were concentrated to 100 μM (1.4 mg/ml) with peptide concentration varying between 0.8 and 5 mM (depending on the peptide). Peptides were titrated into TBC1D23 C-terminal domains with 20 injections of 2.4 μl each separated by 300-s intervals. Experiments were conducted at 20°C. A relevant syringe-peptide-into-buffer blank was subtracted from all data, and for constructs that displayed measurable binding, three independent runs that showed clear saturation of binding were used to calculate the mean KD of the reaction, the stoichiometry (n), and the SEM calculated using NanoAnalyze. Data were exported to GraphPad Prism for figure generation.
6
Peptide-Protein Interaction Kinetics
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ITC experiments were carried out on a Nano ITC machine (TA Instruments) in 100 mM MES (pH 6.8), 150 mM NaCl at 25 °C. Peptides (1–1.5 mM) were titrated onto proteins (100–200 μM) in a 300-μL sample cell while stirring at 200 rpm. The data (Figure S2 ) were analyzed with NanoAnalyze (v3.6.0, TA Instruments) and the KD values were calculated from a single replicate using a single-binding site model.
7
Binding interactions of Hexamer-2 and NUP153
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Binding reactions were performed in a TA Instruments NanoITC machine at 25 °C in a 25 mM Tris, 75 mM NaCl, pH 8 buffer. Hexamer-2 was stable in these conditions after overnight dialysis and during experiments. Synthesized NUP153 peptide (Genscript Co.) was resuspended in the same buffer and injected as the titrant to the Hexamer-2 in the cell. Data were analyzed using the NanoAnalyze (TA Instruments) software, and curves were fitted with an independent one-site binding model.
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