Kinetic analysis of a protein-protein complex to determine its dissociation constant (kd) and the effective concentration (ec50) of an interplaying effector molecule using bio-layer interferometry

Biolayer interferometry (BLI) is an emerging analytical tool that allows the study of protein complexes in real time to determine protein complex kinetic parameters. This article describes a protocol to determine the KD of a protein complex using a 6×His tagged fusion protein as bait immobilized on the NTA sensor chip of the FortéBio® Octet K2 System (Sartorius). We also describe how to determine the half maximal effective concentration (EC50, also known as IC50 for inhibiting effectors) of a metabolite. The complete protocol allows the determination of protein complex KD and small molecular effector EC50 within 8 h, measured in triplicates.