How do you characterize protein-protein interactions biochemically?

PPI characterization uses biophysical and biochemical methods. Affinity measurement: isothermal titration calorimetry (ITC) provides Kd, stoichiometry, and thermodynamics in solution; surface plasmon resonance (SPR) gives kinetics (kon, koff) and Kd; microscale thermophoresis (MST) requires minimal sample. Structural characterization: X-ray crystallography of complexes, cryo-EM, NMR (for smaller complexes), and hydrogen-deuterium exchange MS for interaction interfaces. Functional characterization: pull-down assays, co-immunoprecipitation, and competition experiments. Mapping interaction sites: cross-linking mass spectrometry (XL-MS), alanine scanning mutagenesis, and peptide arrays. In-cell methods: FRET/BRET for live-cell interactions, proximity ligation assays. Integration identifies binding sites, determines affinity, and provides mechanistic understanding for therapeutic targeting.