Biochemistry Unit

Protein Analysis

Biophysical characterization of proteins and antibodies

The facility can provide a precise analysis of the biophysical features related to single proteins and to the interactions among proteins and proteins/substrate or specific antibodies.

The following techniques are available:

Isothermal Titration Calorimetry (VP-ITC): it is the most quantitative means for measuring the thermodynamic properties of biological macromolecule interactions in solution. In particular, ITC determines the binding equilibrium directly by measuring the heat evolved or adsorbed on association of a ligand with its binding partner. In a single experiment, the values of the binding constant (Ka), the stoichiometry (n) and the entalphy of binding (DeltaHb) are determined.

Circular Dichroism (CD): it is a spectroscopic technique that allows obtaining information on protein secondary and tertiary structures.

Fluorescence Spectroscopy: protein intrinsic fluorescence could give information on tertiary structure changes (folding and unfolding) and aggregation (aggregation index). Extrinsic fluorescence of specific dyes allows the study of protein conformational changes. FRET (Fluorescence Resonance Energy Transfer) in vitro analysis can provide information on molecule interactions.

Dynamic Light Scattering (DLS): it gives information on the hydrodynamic properties of macromolecules. In particular, DLS is used to characterize macromolecular oligomerization and aggregation.

Surface Plasmon Resonance (BIAcore 2000): it allows visualizing the interaction process as a function of time between a ligand immobilized on the surface of a sensor chip and an analyte in solution. In particular, SPR provides quantitative information on binding specificity, kinetics, and affinity.