**4. Determination of binding constants**

As discuss above the binding constant provides important and interesting information about the system studied. We will present a few of the multiple experimental posibilities to measure this constant (further information could be found in the literature (Johnson *et al.* 1960; Connors 1987; Hirose 2001; Connors&Mecozzi 2010; Pollard 2010)). It is essencial to keep in mind some crucial details to be sure to calculate the constants properly: it is important to control the temperature, to be sure that the system has reached the equilibrium and to use the correct equilibrium model. One common mistake that should be avoid is confuse the total and free concentrations in the equilibrium expression.

Different techniques are commonly used to study the binding of ligands to their targets. These techniques can be classified as calorimetry, spectroscopy and hydrodynamic methods. Hydrodynamic techniques are tipically separation methodologies such as different chromatographies, ultracentrifugation or equilibrium dialysis with which free ligand, free target and complex are physically separated from each other at equilibrium, thus concentrations of each can be measured. Spectroscopic methodologies include optical spectroscopy (e.g. absorbance, fluorescence), nuclear magnetic resonance or surface plasmon resonance. Calorimetry includes isothermal titration and differential scanning. Calorimetry and spectroscopy methods allow accurately determination of thermodynamics and kinetics of the binding, as well as can give information about the structure of binding sites.

Once the bound (or free) ligand concentration is measured, the binding proportion can be calculated. Other thermodynamic parameters can be calculated by varying ligand or target concentrations or the temperature of the system.
