Bio-Microcalorimetry enables the thermodynamic characterization of  macromolecules like  proteins, nucleic acids, antibodies, lipids, oligosaccharides and many others, in solution.

Its special example is Isothermal Titration Calorimetry (ITC), which is dedicated to the study of molecular interactions. ITC is the only biophysical technique that can simultaneously determine all binding parameters: equilibrium constant (Ka), enthalpy (ΔH), entropy (ΔS) and stoichiometry of the reaction, in a single experiment. Interacting components does not require chemical tagging or immobilization.

Core Facility has MicroCal VP-ITC, a highly sensitive calorimeter that allows for analysis of dilute solutions of macromolecules (typically in the µmolar range in case of proteins).

Technical Specifications

• ITC system can directly measure milimolar to nanomolar binding constants (Ka=10³ x M^-1 – 10⁹x M^-1).
• The operating temperature range is of 5°C to 80°C.
• Measuring cell material: Hastelloy
• Experimental cell volume: 1,4 ml (safe loading require 2 ml of the sample)
• Syringe volume: 280  µl (loading 500 µl total)

Sample preparation requirements

• Both, ligand and cell sample should be dissolved in the exactly same buffer .
• If one molecule requires a small percentage of organic solvent for solubility, this same amount is required in  the  other  sample.
• The starting concentration of ligand (in siringe) should be at least 10 times higher than the concentration of macromolecule.
• The macromolecule concentration in the ITC cell is typically determined by the “C parameter” determined by: C= N[M]T/KD,   where [M]T is the macromolecule concentration in the ITC cell, and N is the stoichiometry. To get a reasonable ITC binding isotherm, the optimal range is 5 < C < 500.
  
• Viscous  additives  such  as  glycerol  should  be  kept  at  a  low  concentration  (less than 20%) for better mixing.
• If  the  presence  of  reducing  agent  is  required  for  a  protein  stability,  use rather low concentration (1 mM) of TCEP or ß-mercaptoethanol  than DTT.
• Buffers with a  large  enthalpy  of  ionization, like TRIS, are not recommended.

Reading

VP-ITC MicroCalorimeter User’s Manual