Calorimetry - Instruct-CZ

VP-DSC calorimeter measures heat changes that occur in the sample (biomolecule solution) during a controlled increase or decrease in temperature, on the basis of a temperature difference between the sample and the reference material. It is a valuable technique for the study of samples in solution providing fast and accurate determination of the transition midpoint Tm - when 50 % of the biomolecule are unfolded. In a typical arrangement of ITC (Isothermal Titration Calorimetry), the titrant, also referred as the ligand, is injected into the sample cell containing the macromolecule sample solution. The calorimetric measurement can be done over a range of biologically relevant conditions (temperature, salt, pH, etc.).

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User Guide

Differential Scanning Calorimetry (DSC)

  • VP-DSC – differential scanning microcalorimeter – measures heat changes that occur in the sample (biomolecule solution) during a controlled increase or decrease in temperature, on the basis of a temperature difference between the sample and the reference material.
  • It is a valuable technique for the study of samples in solution providing fast and accurate determination of the transition midpoint Tm – when 50% of the biomolecule are unfolded.
  • In addition, a complete thermodynamic profile is generated to understand the factors that affect conformation and stability - enthalpy (ΔH) of unfolding due to heat denaturation, also the change in heat capacity (ΔCp) of denaturation can be determined.
  • DSC is a sensitive, easy-to-use technique that requires no assay development, labelling or immobilization. Filling of the cell is crucial for the accuracy.
  • The operating temperature range is of -10°C to 130°C.
  • Scanrates fall in the range of 0°C/hr to 90°C/hr in the upscan mode and 0°C/hr to -60°C/hr in the downscan mode. Experiment at constant temperature (Isoscan) for shelf life studies or evaluating of the compound stability is also possible.

DSC can be used:
  • for characterization of the stability of proteins or other biomolecules, for elucidation the factors that contribute to the folding and stability of native biomolecules, including hydrophobic interactions, hydrogen bonding, conformational entropy, and the physical environment.
  • for characterization of membranes, lipids, nucleic acids and micellar systems. Assessment of the effects of structural change on a molecule’s stability - protein engineering or antibody domain studies.

Data collection:
  • Conventional DSC - mode uses a linearly increasing or decreasing temperature ramp function, while measuring the differential.
  • Isothermal Scan Mode – a constant temperature is maintained for a relatively long period of time while measuring the differential power between the reference cell and sample cell.
  • Proper sample preparation is crucial for the successful DSC measurement. Sample buffer and buffer for filling the reference cell should be EXACTLY the same.
  • The sample solutions should be dialysed against the buffer solution used for DSC measurement, if it is possible. The pH should be checked before the measurement.
  • Volume of sample for filling the sample cell and buffer for filling the reference cell must have at least 800.0 µl, typically 1.0 ml is recommended.
  • If the reducing agent is needed in the sample, usage of up to 5 mM b-mercaptoethanol (or TCEP) instead of DTT is recommended.
  • Fluoride compounds can cause irreparable damage of the VP-DSC cell, therefore it is not possible to measure samples containing fluorides.
  • Precipitation and aggregation can cause a rapid downward shift or an increase in baseline noise after the system unfolds. Minimizing precipitation is necessary for accurate result.


Isothermal Titration Calorimetry (ITC)

  • ITC method is used for characterization of biomolecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids and others.
  • Enzyme kinetics, biological activity or the effect of molecular structure changes on binding mechanism can be also assessed.
  • Complete thermodynamic profile of the molecular interaction in a single experiment (stoichiometry, Ka, ∆H and ∆S values) or kinetics parameters Km and kcat can be determined.
  • Auto-iTC200 and VP-ITC are designed to measure the heat of binding. In a typical arrangement, the titrant, also referred as the ligand, is injected into the sample cell containing the macromolecule sample material.
  • The calorimetric measurement can be done over a range of biologically relevant conditions (temperature, salt, pH, etc.).
  • ITC system directly measures submilimolar to nanomolar binding constants (103 - 109 M-1). Interactions with nanomolar to picomolar binding constants (109-1012 M-1) can be measured using the competitive binding technique, the same principle can be used for low affinity interactions (103-102 M-1). The operating temperature range is of 2°C to 80°C.
  • Two operation modes are available: user self-operating measurement (VP-ITC) or performing the measurement by core facility staff (Auto-iTC200).

Established methodologies and provided services:
  • Calorimetric measurement of protein-ligand interaction (Standard titration method, Single injection method)
  • Competitive-based measurement - low or high affinity interactions
  • Data evaluation - thermodynamic parameters determination using curve fitting models: One set of binding site, Two sets of binding sites
  • Eventuality of manual data evaluation using fitting models: Sequential binding sites, Competitive binding, Dissociation

Data processing:
  • Using the Origin software and NITPIC (possibility to train people in data processing)

Sample requirements - importance of sample preparation
  • Proper sample preparation is crucial for the successful ITC measurement. The buffer solution, in which the macromolecule and the ligand are dissolved, should be exactly the same. Concentration of samples must be determined precisely.
  • The macromolecule sample (the sample placed in the cell) must have a volume at least 450.0 µl for Auto-iTC200 and 1800.0 µl for VP-ITC calorimeter. Preferably, the macromolecule solutions should be dialysed against the buffer solution used for the ITC measurement. A lyophilized macromolecule sample devoid of salts or additives may be dissolved directly into the buffer, the pH should be checked before the measurement.
  • The ligand solution (the sample placed in the injection syringe) must have a volume at least 150.0 µl for Auto-iTC200 and 500.0 µl for VP-ITC calorimeter. Generally a concentration of ligand should be 10 times higher than the concentration of macromolecule.
  • High affinity interactions can be studied at low concentrations. In this case the minimum concentration of macromolecule sample which causes measurable heat is 10 µM. For low affinity interactions the macromolecule sample concentration should be 5 times of Kd or higher, but higher concentration may be limited by availability or solubility of samples.
  • Calculating the cell sample concentration - M = c / (n x Ka)
  • c-value … (should lie between 10-500); n … binding stoichiometry;
  • M … molar concentration of the cell sample; Ka …association constant; Kd … dissociation constant
  • At least 10 ml of the used buffer must be sent for each measurement (for Auto-iTC200).
  • If possible, choose a buffer with low ionization heat in order to minimize artificial heats of buffer ionization (e.g. phosphate buffer works well).
  • If the presence of reducing agent is required for a protein stability, then ß‑mercaptoethanol (less than 5 mM) or TCEP (less than 2 mM) should be used rather tan DTT.