Differential scanning calorimetry (DSC) experiments are carried out in 1 mM HEPES buffer at pH 7.4 containing 10 mM NaCl and 2 mM CaCl2 in a Microcal VP-DSC instrument (Malvern, U.K.). Several water–water and buffer–buffer scans are acquired before the protein scan to equilibrate the cells. The protein scans are acquired at a concentration of ∼11.2 μM and ∼15.1 μM for the WT and S47P mutant, respectively, at a scan rate of 1 K/min. The absolute heat capacity is calculated using the method of Sanchez-Ruiz and co-workers [30 (link)].
Vp dsc instrument
Manufactured by Malvern Panalytical
Sourced in United Kingdom, United States
The VP-DSC instrument is a differential scanning calorimeter (DSC) designed and manufactured by Malvern Panalytical. It is a thermal analysis instrument used to measure the heat flow associated with material transitions and reactions as a function of temperature and time.
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Lab products found in correlation
Microcal origin software, by Malvern Panalytical (2 mentions)
Origin 7, by OriginLab (2 mentions)
Microcal origin 7, by Malvern Panalytical (2 mentions)
Origin 7, by Malvern Panalytical (2 mentions)
Farnesol, by Merck Group (1 mentions)
Cm5 chip, by GE Healthcare (1 mentions)
Human fh, by Complement Technology (1 mentions)
Biacore x100 plus instrument, by GE Healthcare (1 mentions)
Origin software, by OriginLab (1 mentions)
Cary 300 bio uv vis spectrophotometer, by Agilent Technologies (1 mentions)
22 protocols using vp dsc instrument
1
DSC analysis of protein thermal stability
2
Reversible ELP Phase Transition Analysis
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All DSC experiments
were performed using a Microcal (Northampton, MA) VP-DSC instrument.
For all experiments, both the sample and reference cells were filled
with the appropriate buffer for baseline calibration, and the sample
cell was refilled with the appropriate ELP solution. The protocol
was set to ramp the temperature from 283 to 343 K (10–70 °C)
at a rate of 1 °C/min and then rapidly cool the solution to 10
°C over 5 min. This was repeated twice to examine the reversibility
of the ELP phase transition. Enthalpy ΔH was
calculated by integrating the baseline-corrected excess heat capacity
signal from 10 °C on either side of the phase change.
were performed using a Microcal (Northampton, MA) VP-DSC instrument.
For all experiments, both the sample and reference cells were filled
with the appropriate buffer for baseline calibration, and the sample
cell was refilled with the appropriate ELP solution. The protocol
was set to ramp the temperature from 283 to 343 K (10–70 °C)
at a rate of 1 °C/min and then rapidly cool the solution to 10
°C over 5 min. This was repeated twice to examine the reversibility
of the ELP phase transition. Enthalpy ΔH was
calculated by integrating the baseline-corrected excess heat capacity
signal from 10 °C on either side of the phase change.
3
Differential Scanning Calorimetry of Protein-Ligand Interactions
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DSC experiments were carried out using a Microcal VP-DSC instrument with protein concentration of 10 μM at a scan rate of 30 °C/h. Samples were degassed extensively under vacuum before each experiment. Scans were preformed from 30 °C to 75 °C, then immediately cooled back to 30 °C and held for 10 min for equilibration and a second scan (rescan) was carried out at the same scan rate as the control. These scans were performed in 50 mM phosphate buffer pH 7.4, in the absence and presence of 0.5 mM NADP+. Prior to data analysis, the control (rescan data) was subtracted from the experimental data, followed by data normalization for protein concentration. Deconvolution analysis35 (link) and plotting of the calorimetric data was performed using Origin software (MicroCal).
4
Differential Scanning Calorimetry of DNA-Ligand Interactions
5
Differential Scanning Calorimetry in PBS
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DSC experiments were carried out in PBS as described previously49 (link) using a VP-DSC instrument (Microcal, Northampton, MA) and data was analyzed with the software supplied with the equipment.
6
Differential Scanning Calorimetry of Protein-Peptide Interactions
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Experiments were carried out in a VP-DSC instrument from Microcal INC. as described elsewhere [11 (link)]. The buffer conditions were the same as with ITC experiments, 50 mM buffer potassium phosphate at a pH of 7.5. For experiments including the peptides, the final concentration ratio was 1:3 (protein:peptide). Final data were obtained after treatment with ORIGIN 7.0 (OriginLab Corporation, Northampton, MA, USA, 2002).
7
Measuring 14-3-3ζ C94 Mutant Thermal Stability
8
DSC Analysis of Protein-Lipid Interactions
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Differential scanning calorimetry (DSC) was carried out essentially as earlier described for myelin basic protein (MBP) [36 (link)]. P2 and P2 F57A were mixed with MLVs of 350 μM DMPC:DMPG (1:1) in 20 mM HEPES, 150 mM NaCl, pH 7.5, at 1:100 and 1:200 protein-to-lipid (P/L) ratios. The samples were degassed for 10 min under vacuum with stirring at + 10 °C before measurements. A control lipid sample without protein was also prepared.
DSC measurements were performed with a MicroCal VP-DSC instrument. Cell volume was 527.4 μl. The reference cell was filled with 20 mM HEPES, 150 mM NaCl, pH 7.5. Each sample was scanned from + 10 to + 40 °C and back to + 10 °C in 1 °C/min steps. Buffer baselines were subtracted from sample curves and zeroed between + 28–30 °C. All samples were prepared and measured twice and deemed reproducible.
DSC measurements were performed with a MicroCal VP-DSC instrument. Cell volume was 527.4 μl. The reference cell was filled with 20 mM HEPES, 150 mM NaCl, pH 7.5. Each sample was scanned from + 10 to + 40 °C and back to + 10 °C in 1 °C/min steps. Buffer baselines were subtracted from sample curves and zeroed between + 28–30 °C. All samples were prepared and measured twice and deemed reproducible.
9
Calorimetric Analysis of Protein Unfolding
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For the calorimetric study of protein unfolding, the protein samples were dialyzed overnight against a buffer containing 50 mM sodium phosphate and 5% w/v glycerol at pH 7.2 and pH 8, which was also used in the reference cell and for base line determination. The final protein concentrations were 1 mg mL−1. Heat absorbance was collected from 20 – 55°C in a MicroCal VP-DSC instrument at a temperature ramp rate of 1°C min−1. Thermograms were base-line corrected, normalized, and analyzed according to two-state and non-two-state models with a single and multiple transitions, in which Tm, ΔHcal, and ΔHvH, and ΔCp of individual transitions were fitted independently using the MicroCal Origin software (Microcal, Northhampton, MA).
10
Calorimetric Analysis of Protein Unfolding
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