A Familiar Protein-Ligand Interaction Revisited with Multiple Methods

The interaction of hen egg white lysozyme with the trisaccharide tri-N-acetyl glucosamine has been well-characterized by biophysical methods and structural biology. In this chapter, we present a series of experiments designed to detect and quantify that interaction using several commonly available biophysical methods: thermal shift assay, fluorescence intensity, microscale thermophoresis, isothermal titration calorimetry, and surface plasmon resonance.These experiments have been used for teaching and troubleshooting in a core facility. By taking a set of representative data from several years of practical courses, we are able to demonstrate the robustness of the protocols, calculate confidence intervals for the dissociation constant from each method, and illustrate the degree of consistency between those methods when applied to a simple system in a single location by different experimenters.