A TA Instruments Nano ITC Low Volume isothermal titration calorimeter was used. The excess ligand in the original NP aqueous solution was removed by centrifugation at 1500 rpm for 3 min and the NPs were then redispersed in water. Both d - and l -Cys CdTe NPs had the same size (3.2 nm), concentration (0.02 mM), and pH value (9.9). The NP solution in the sample cell and the syringe had the same concentration (0.02 mM). The syringe volume was 50 μl, with 2.5 μl per injection (20 injections in total) and an injection interval of 150 s. An initial baseline was collected for 100 s before the first injection. The temperature was set to 22°C. The stirring rate was 350 rpm. NanoAnalyze software was used to analyze the raw heat rate graphs and to model with a constant blank model and an independent model.
Nano itc low volume isothermal titration calorimeter
Manufactured by TA Instruments
Sourced in United States
The Nano ITC Low Volume isothermal titration calorimeter is a lab equipment designed to measure the heat effects of interactions between molecules. It can quantify the thermodynamic parameters of a wide range of chemical and biological binding events.
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5 protocols using nano itc low volume isothermal titration calorimeter
1
Isothermal Titration Calorimetry of Cysteine-Capped CdTe Nanoparticles
2
Characterizing vMIP-II Binding to vCCI
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A Nano ITC Low Volume isothermal titration calorimeter (TA Instruments, New Castle, DE, USA) was loaded with degassed 10 uM vCCI in 20 mM NaOP, 100 mM NaCl, pH 7.0 and water in the reference cell. Twenty 2.5 μL injections of 100 μM vMIP-II also in 20 mM NaOP, 100 mM NaCl, pH 7.0 were then injected at 300 second intervals, with a 350 rpm stirring speed. Baseline selection, buffer-into-buffer blank was subtracted from the data, and peak-by-peak manual integration was performed using NanoAnalyze software (TA Instruments, New Castle, DE, USA). The data for an independent binding site model was provided by the software. The Kd was below detectable limits (10−10 nM).
3
Isothermal Titration Calorimetry of Metal Oxyanion Binding
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Experiments were performed on a Nano ITC Low Volume isothermal titration calorimeter (TA Instruments, United States) at a temperature of 25°C. Forty micromolar metal-free ModA was loaded into the sample cell of the calorimeter and titrated with 320 μM of metal oxyanion ligand solutions (K2CrO4, Na2MoO4, and Na2WO4). An initial injection of 1 μl of ligand solution was followed by a series of 2 μl titrations with an injection interval of 200 s for a total of 25 cycles. Data were analyzed using the NanoAnalyze Data Analysis software (v3.10.0, TA Instruments, United States). Enthalpy changes were corrected by subtracting the background mean enthalpies from the raw titration data and data were then normalized with the ligand concentrations. Excluding the first data point, an “independent” model for a single binding site for the ligands was used to fit the data.
4
Measuring T4 Ligase Loop Peptide-gp45 Binding
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ITC was performed to determine the dissociation constant (Kd) for binding of the T4 ligase loop peptide to gp45. Purified gp45 was dialyzed against 0.2 M NaCl, 10 mM Tris–HCl pH 7.4, and 10 mM β-mercaptoethanol at 4°C in a 3.5 kDa cutoff membrane. An HPLC-purified loop peptide KKEPEGLDFLFDA (>95% pure, Selleck Chemicals) was dialyzed against the same buffer in a 2 kDa cutoff Slide-A-lyzer cassette (Thermo Fisher). Peptide and gp45 were adjusted to the desired concentrations with used dialysis buffer as measured by A257 (ϵ = 220 M−1 cm−1) and A280 (ϵ = 19 940 M−1 cm−1), respectively. ITC was performed on a Nano ITC Low Volume isothermal titration calorimeter (TA Instruments). ITC was run at 25°C with 300 rpm stirring. The instrument was equilibrated for 30 min to reach a stable baseline. After a small injection of 0.49 μl to remove buffer that had diffused into the needle, 2.49 μl of loop peptide (525 μM) was injected into the experimental cell with an initial 300 μl gp45 (51 μM) every 5 min for 20 injections. The reference cell was always filled with water. Four replicates were performed as described above, and a blank experiment was performed with loop peptide injected into used dialysis buffer. A fifth experiment was performed with 100 μM gp45 in the experimental cell. The data were analyzed in the NanoAnalyze software (TA Instruments).
5
Peptide Binding Kinetics with Rho Protein
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Binding characteristics of peptide 33 and peptide 16 with Rho P167L protein were examined with ITC using a Nano-ITC Low-Volume isothermal titration calorimeter (TA Instruments). 1 μM Rho P167L in the ITC buffer (25 mM Tris Cl, pH 8.0, 50 mM KCl, 100 mM NaCl, 5 mM MgCl2, 1 mM ATP, 0.01% Triton X-100, and 5% glycerol) was titrated against 50 μM peptide solution prepared in the same buffer. ITC experiments were performed at 25 °C by injecting 2.5-μl injections of peptide solution into the Rho solution with constant stirring at 200 rpm. To identify the probable binding region(s) of peptide 33 on the Rho protein, ITC was performed in the presence of 10 μM Poly(dC)34, 2 mM Poly(rC), or 5 μM NusG-CTD premixed with the Rho protein under the same conditions described above. The thermodynamic parameters of all the interactions were obtained using the in-built software of the calorimeter. The specific buffer composition in these experiments was chosen to minimize the heat of dilution and noise and to reduce aggregation during a slow stirring inside the ITC cells used during the injections of the peptides.
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