Best practices for isothermal titration calorimetry of protein-DNA interactions.

Isothermal titration calorimetry (ITC) is a powerful label-free method for quantifying protein-DNA interactions, providing direct measurements of binding affinity, stoichiometry, enthalpy, and entropy in a single experiment. However, the quality of protein-DNA ITC data depends strongly on experimental details, including accurate concentration determination, careful buffer matching, sample quality, instrument cleanliness, sample loading, degassing, and calibration. Without careful control, these factors can obscure true binding behavior and complicate thermodynamic interpretation. Here, we present a practical checklist for designing, optimizing, and troubleshooting ITC experiments for protein-DNA binding studies. Using homeodomain transcription factor-DNA interactions as representative examples, we identify common failure modes, including concentration errors, buffer mismatch, poorly optimized binding-partner concentrations, sample carryover, cell or syringe contamination, loading artifacts, and calibration problems. For each potential issue, we outline how to recognize the problem, choose appropriate controls, and apply corrective strategies to improve data quality and reproducibility. Together, this checklist provides a practical framework for improving protein-DNA ITC experiments by helping researchers distinguish genuine binding behavior from correctable technical artifacts and generate more reliable thermodynamic measurements.