Analysis of interactions by calorimetry (ITC)
ITC is the most precise technique to measure the affinity between macromolecules such as proteins, nucleic acids, or any small molecule.
In a typical experiment (e.g. protein-peptide) aliquots of a titrant (peptide in the syringe) are injected into the protein solution (in the sample cell).
Upon each titration the amount of heat released or absorbed (proportional to the complex formed) is measured and used to determine the equilibrium dissociation constant (Kd), the number of binding sites or stoichiometry (n) and the binding enthalpy (ΔH) and entropy (ΔS) ─ Figure 2.
The last two terms contain information about the type of interaction and reflect the real nature of the forces driving the binding.
ITC is an equilibrium solution technique and is truly the only direct method to quantify Kd values without the use any label. ITC is relatively artefacts-free as is not affected by the optical properties of the samples and is not limited by the ligand or protein sizes.
A direct experiment in the ITC200 instrument detects affinities ranging from nanomolar to low millimolar, although an indirect experiment (competition with a ligand of known affinity) can be used in to extend from pico- to sub-millimolar.
Applications and Booking
Characterization of molecular interactions established by proteins, nucleic acids, antibodies, lipids, sugars, etc. and any small molecules (peptides, compounds, metals, etc.).
Assessment of biological activity.
Assessment of the effect of molecular structure changes on binding mechanisms, dimerization, etc.
EMBL Grenoble provides research services such as access to third-generation synchrotron beamlines for macromolecular crystallography at the ESRF, and instruments for neutron crystallography on the high-flux neutron source of the ILL.