Biophysical Characterization of
Protein:Ligand Interactions

Biophysical methods such as TSA, SPR, or ITC are orthogonal to commonly used high-throughput screening methods and can provide confirmation of the molecular interaction of HTS hits. Each method provides different data to guide discovery and development.


  • Measures the temperature at which a protein unfolds (Tm)
  • Measures ligand-induced changes to thermal stability
  • Ranks ligands by affinity; is powerful method for fragment-based screening
  • Maps protein stability in various buffer conditions
  • Has minimal protein requirements
  • Can screen 10,000 compound per week

Surface Plasmon Resonance (SPR)

  • Measures association and dissociation rates (Ka and Kd); enables calculation of the equilibrium dissociation constant (KD)
  • Elucidates the interaction kinetics between a protein and its ligand
  • Requires attachment of the protein or the ligand to a biosensor chip

Isothermal Titration Calorimetry (ITC)

  • Provides a complete thermodynamic profile of protein:ligand interactions
  • Protein and ligand remain  free of modification, tags, or coupling
  • Quantitatively determines equilibrium binding affinity (Ka), binding stoichiometry, and enthalpy of binding (ΔH)
  • Can determine entropy (ΔS), free energy of binding (ΔG), and dissociation constant (Kd) by extension