ITC measurements were performed at 25 °C using a MicroCal auto-iTC200 calorimeter (Malvern Panalytical). Apo DnaK T175A and NBD T175A were purified as described, with a final dialysis step against 40 mM Tris-HCl (pH = 7.4), 50 mM KCl, 10 mM MgCl2 for 16 h at 4 °C. The concentration of the proteins after dialysis was measured using a DC protein assay. For the ITC assay, the dialyzed proteins were prediluted to 60 μM in the dialysis buffer, then diluted 1:1 with 2X buffer to a final concentration of 30 μM in a final buffer containing 40 mM Tris-HCl (pH = 7.4), 50 mM KCl, 10 mM MgCl2 supplemented with 1% DMSO and 0.01% tween-20. Then, 19 injections of 2 μL telaprevir or MECA (100 μM), dissolved in the aforementioned buffer, were injected into 0.2 mL of either DNAK175A or NBD in the chamber every 150 s. For one replicate, telaprevir was dissolved in 40 mM Tris-HCl (pH = 7.4), 50 mM KCl, 10 mM MgCl2 supplemented with 1.5% DMSO and 0.015% tween-20. Data for raw ITC and thermodynamic curves were downloaded for analysis using Affinimeter software and plotted using GraphPad Prism. Heat release from dilution of drug into buffer was subtracted from the thermodynamic data.
Microcal auto itc200 calorimeter
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The MicroCal auto-iTC200 calorimeter is a laboratory instrument designed for performing isothermal titration calorimetry (ITC) measurements. It is capable of accurately measuring the heat effects associated with molecular interactions, such as protein-ligand binding, enzyme-substrate reactions, and other biomolecular processes.
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9 protocols using microcal auto itc200 calorimeter
1
ITC Analysis of DnaK Protein Interactions
2
Isothermal Titration Calorimetry of IL-17A and IL-17RA Interaction
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ITC was performed with a MicroCal Auto-ITC200 calorimeter (Malvern Instruments, UK) at 298K. Proteins were dialyzed overnight against the same reaction buffer (PBS pH 7.4, 1mM EDTA). Protein concentration was quantified by monitoring UV absorbance at 210nm using High-Performance Liquid Chromatography (HPLC). All titrations were made by 26 successive 3s injections of 1.5μL cytokines into 120μL of IL-17RA solution with a 180s interval between consecutive injections. An initial delay of 60s was applied before the first injection and a stirring speed of 750rpm was applied. For the wild-type receptor, the experiment was performed with 250μM IL-17A (syringe) and 35μM IL-17RA (cell). For the experiment with the A104E receptor variant, 149μM IL-17A and 22μM A104E IL-17RA were used. The binding isotherms were fitted to a single-site binding model under the MicroCal PEAQ-ITC analysis software (version 1.21, Malvern Instruments, UK).
3
Thermodynamic Characterization of cGAS-Ligand Interactions
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ITC measurements were performed at 25 °C using a MicroCal auto-iTC200 calorimeter (MicroCal, LLC). Purified truncated mouse cGAS was dialyzed against 40 mM HEPES buffer (pH 7.5) and 300 mM NaCl for 24 h at 4 °C. Dialysis led to some precipitation, which were removed through centrifugation followed by collection of the supernatant. The concentration of the protein in supernatant was measured using Pierce® BCA protein assay. The protein was flash frozen in liquid N2 and stored at −80 °C. For the ITC assay, the dialyzed protein was prediluted to 20 µM in the dialysis buffer and diluted to a final concentration of 10 µM in a final buffer containing 40 mM HEPES, 1% DMSO, 150 mM NaCl, 0.01% Tween-20 and + /− dsDNA 10 µM and + /− ATP or GTP 1 mM. Then, 2 μl RU.365 100 µM, dissolved in the buffer aforementioned, were injected into 0.2 ml of protein in the chamber every 150 s. Data for raw ITC and thermodynamic curves, each from one experiment, were downloaded after analysis using Affinimeter software and plotted using GraphPad Prism (7.01). The software calculates error bars as an indication of the contribution of the raw data noise that is determined from the integral of the standard deviation of the baseline during the injection.
4
Isothermal Titration Calorimetry Measurements
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ITC measurements were performed at 15°C using a MicroCal auto-iTC200 calorimeter (MicroCal, LLC). Protein samples were extensively dialyzed against a buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.5 mM TCEP, and 1% DMSO. Typically 5 μL of 2.0 mM protein was injected into 0.4 mL of 0.2 mM ligand in the chamber every 150 s. Baseline-corrected data were analyzed with ORIGIN software.
5
Thermodynamic Analysis of TTR Ligand Binding
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The thermodynamic
parameters that characterize the binding of TTR ligands to WT-TTR
were determined using a MicroCal Auto-iTC200 Calorimeter (MicroCal,
Malvern-Panalytical), as detailed before.44 (link) A 100 μM solution of the compound (in a PBS buffer pH 7.0
containing 100 mM KCl, 1 mM EDTA and 2.5% DMSO) was titrated into
an ITC cell containing a 5 μM solution of WT-TTR in the same
buffer at 25 °C. A stirring speed of 750 rpm and 2 μL injections
were programmed, with a 150 s equilibration period between each injection
to allow the calorimetric signal (thermal power) to return to baseline
and a 10 μcal/s reference power. Two independent titrations
were done for each TTR ligand. The experimental data were analyzed
with a general model for a protein with two ligand-binding sites93 (link),94 (link) implemented in Origin 7.0 (OriginLab) accounting for cooperative
and noncooperative binding. The best fit of the binding isotherm was
attained with a model considering two identical binding sites (i.e.,
no cooperativity) for tolcapone and its derivatives.
parameters that characterize the binding of TTR ligands to WT-TTR
were determined using a MicroCal Auto-iTC200 Calorimeter (MicroCal,
Malvern-Panalytical), as detailed before.44 (link) A 100 μM solution of the compound (in a PBS buffer pH 7.0
containing 100 mM KCl, 1 mM EDTA and 2.5% DMSO) was titrated into
an ITC cell containing a 5 μM solution of WT-TTR in the same
buffer at 25 °C. A stirring speed of 750 rpm and 2 μL injections
were programmed, with a 150 s equilibration period between each injection
to allow the calorimetric signal (thermal power) to return to baseline
and a 10 μcal/s reference power. Two independent titrations
were done for each TTR ligand. The experimental data were analyzed
with a general model for a protein with two ligand-binding sites93 (link),94 (link) implemented in Origin 7.0 (OriginLab) accounting for cooperative
and noncooperative binding. The best fit of the binding isotherm was
attained with a model considering two identical binding sites (i.e.,
no cooperativity) for tolcapone and its derivatives.
6
Calorimetric Analysis of PpBMT/COMT–SAH Interactions
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The equilibrium dissociation constants of PpBMT/COMT–SAH interactions were determined by an MicroCal Auto-ITC200 calorimeter (MicroCal, Malvern, UK). The binding protein PpBMT/COMT (50–100 μM) and cofactor analogue SAH (500 μM) were measured in the buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl) at 25 °C. ITC data were analyzed and fitted in Origin 7 (OriginLab, Northampton, MA, USA) to determine the site-binding model that produced a good fit for the resulting data.
7
Isothermal Titration Calorimetry of ENZ-PRO
8
Isothermal Titration Calorimetry of Katanin-p60
9
Isothermal Titration Calorimetry of OTUD7A
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ITC measurements were performed using a MicroCal auto‐iTC200 calorimeter (MicroCal, LLC) as described previously.[61 ] Briefly, His‐tagged OTU domain of OTUD7A proteins were purified using BL21 strain upon induced by 0.3 × 10−3m isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) when OD600 around 0.6 overnight at 16 °C. Then, purified human His‐tagged OTU domain of OTUD7A was dialyzed against 50 × 10−3m HEPES buffer (pH 7.2) with 100 × 10−3m NaCl for overnight at 4 °C. The concentration of the protein was determined by band intensity in a gel‐cod staining SDS‐PAGE gel. The ITC assay was carried out at 37 °C. The dialyzed His‐OTUD7A‐OTU proteins were diluted to 40 × 10−6m in the dialysis buffer containing 4% DMSO. Then, 400 × 10−6m 7Ai, dissolved in the same dialysis buffer with 4% DMSO (50 × 10−3m HEPES buffer (pH 7.2) with 100 × 10−3m NaCl) was injected into 0.4 mL of His‐OTUD7A‐OTU protein in the chamber for every 180 s. The dissociation constants and thermodynamic parameters were determined by using the embedded software package Origin7 (Microcal).
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