VP-ITC MicroCal/Malvern Microcalorimeter
Contact: Roman Szczepanowski
Access to the instrument after short training
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).
- 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.
VP-ITC MicroCalorimeter User’s Manual