Isothermal Titration Calorimetry – ITC

ITC is a particularly suitable and powerful method to study the thermodynamics of protein:protein and protein:ligand interactions. ITC measures thechange in heat upon interaction between two molecules (Δct). The experimental setup is quite simple: A syringe containing a "ligand" (either a small molecule ligand or an interacting protein) is titrated into a highly temperature controlled cell containing the protein solution. Critically, the buffers of both solutions need to be identical, which can be achieved by dialysis of both substances against the same buffer. When the two proteins or the protein and the ligand interact, heat is released or absorbed. When the protein in the cell becomes saturated with added ligand or protein, the heat signal will be come smaller and smaller until only the background heat of dilution is observed. This raw ITC data can be fitted and ΔH and ΔS can be extracted.

It is critical to optimize the starting concentration of the protein in the cell according to the expected dissociation constant. Furthermore, the titrant needs to be reasonably soluble, since an approximately 10 times higher concentration than for the protein in the cell is required. Lastly, the cell requires at least 1.5ml of protein solution and the syringe about 300μl.