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13 Conclusion ITC is a powerful and insightful tool for affinity measurements, especially valued for its ability to deliver comprehensive thermodynamic information without invasive techniques. While its higher sample requirements and lower throughput are notable drawbacks, the quality of information it provides ensures its continued relevance. Isothermal Titration Calorimetry Isothermal titration calorimetry (ITC) is a label-free technique that directly measures the heat changes accompanying biomolecular interactions. How it works During an ITC experiment, one binding partner (the titrant) is gradually injected into a chamber containing the other partner (the analyte), under constant temperature conditions. Each injection of the titrant induces a binding interaction that either absorbs or releases heat, depending on the nature of the reaction. The instrument detects these subtle temperature shi s in real time. This heat profile is then analyzed to yield a complete thermodynamic characterization of the interaction. Strengths ITC offers several notable advantages for studying biomolecular interactions. One of its primary strengths is its ability to provide a full thermodynamic profile including key parameters like binding affinity (K d ), stoichiometry, enthalpy (ΔH), and entropy (ΔS), all within a single experiment. Importantly, ITC does not require any form of labeling, modification, or immobilization of the molecules involved, allowing researchers to study interactions in their native state. This non-invasive approach is particularly advantageous for fragile systems or for studying interactions that might be altered by structural modifications. Weaknesses One major drawback of ITC is its relatively high sample requirement, as it typically necessitates milligram quantities of both the titrant and analyte. This high consumption can be prohibitive, particularly when studying rare, expensive, or difficult-to-purify biomolecules. Furthermore, weak interactions with K d s in the millimolar range o en generate minimal heat, resulting in data with low signal-to-noise ratios that can be challenging to interpret. For multistep binding mechanisms or systems with multiple binding sites the interpretation of ITC data can be complex, as a more advanced analysis may be required to deconvolute overlapping thermodynamic parameters. Additionally, ITC is also relatively low in throughput; each experiment typically spans an hour or more, and the need to clean, reload, and recalibrate between runs can further extend the time required for each interaction study. This limited throughput can make ITC less practical for screening large interaction libraries or for projects where rapid data collection is essential. Furthermore, precise calibration and baseline stability are critical, as even small fluctuations can introduce errors, particularly when measuring low enthalpy interactions.