Our Laboratory Testing Blog

Alternative Approaches for Determining Protein-Ligand Binding Constants

An alternative approach to label-free determination of protein binding strengths with small molecular ligands is Surface Plasmon Resonance (SPR).  SPR is a method for characterizing macromolecular interactions.  It is an optical technique that uses the evanescent wave phenomenon to measure changes in the refractive index very close to a sensor surface.  The binding between an analyte in solution with a ligand on the sensor surface results in a change in the refractive index.  The interaction is monitored in real time and the amount of bound ligand and rates of association and dissociation can be measured with high precision.  Analysis of kinetics and thermodynamics by SPR can be used to understand the complex mechanisms of molecular recognition events.  The great advantage of SPR is the sensitivity and easy access to kinetic data of the non-covalent binding processes.

However, a disadvantage for conventionally SPR-based measurement is that no stoichiometric information is available, and that the process of immobilization is sometimes delicate and often demands lengthy optimization.  It is crucial to minimize or to completely avoid non-specific surface–analyte interactions during the binding experiments.  The sensitivity of an SPR instrument can be a problem since the angle of reflection is detected which is proportional to the mass of the analytes and can generate poor signals for small molecules.  Other disadvantages of SPR have been described in the research article "Label-free determination of protein-ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry" written by Matthias C. Jecklin, Stefan Schauer, Christoph E. Dumelin and Renato Zenobi.