Isothermal Titration
Isothermal Titration - Related Videos
08:45
Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity
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Cited by 84 •
2011
A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.
10:04
Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms
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Cited by 3 •
2011
ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.
13:00
Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions
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Cited by 16 •
2014
Isothermal titration calorimetry measures heat flow released or absorbed in chemical reactions. This method can be used to quantify enzyme-catalysis. In this paper, the protocol for instrumental setup, experiment running, and data analysis is generally described, and applied to the characterization of enzymatic urea hydrolysis by jack bean urease.
08:37
Measuring Enzymatic Stability by Isothermal Titration Calorimetry
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Cited by 1 •
2019
The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme activity.
06:02
Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry
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Cited by 1 •
2022
The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.
FAQs
These parameters provide complementary views of a molecular interaction. Affinity indicates how strongly the partners associate, while stoichiometry describes their interaction ratio. Enthalpy represents heat released or absorbed during recognition, and entropy captures the disorder-related contribution to the interaction. Considering all four measurements helps researchers distinguish and compare binding behaviors rather than relying on a single value.
Each injection produces a separate heat response as the added partner interacts with the substance in the sample. Tracking these responses step by step allows the interaction to be quantified across the titration rather than assessed from one mixing event. The resulting measurements support determination of binding properties and reveal how the interaction changes as available binding capacity is progressively engaged.
Stoichiometry indicates the proportion in which interacting molecules associate. In cancer research, this information can clarify how tumor-associated proteins, signaling molecules, antibodies, or candidate drugs participate in molecular recognition. Combined with affinity and thermodynamic measurements, the interaction ratio helps researchers characterize the molecular basis of recognition and compare how different partners engage the same cancer-related target.
The experiment begins by placing one interaction partner in the instrument’s sample solution and preparing the other for stepwise injection. The instrument maintains a constant temperature while each portion is mixed with the sample and records the resulting heat change. Researchers then use the accumulated injection measurements to determine interaction properties such as affinity, stoichiometry, enthalpy, and entropy.
Researchers can apply the technique when they need quantitative information about interactions involving tumor-associated proteins, signaling molecules, antibodies, or candidate drugs. It is especially useful for examining molecular recognition within cancer-related pathways. The measurements provide a way to characterize these interactions directly and can help clarify how therapeutic molecules engage targets associated with tumor biology.
By measuring the interaction properties of different candidate inhibitors, researchers can compare their binding affinity, stoichiometry, enthalpic contribution, and entropic contribution. These distinctions provide more detailed information than identifying interaction alone. The results can clarify why candidates bind differently and guide the design and optimization of therapeutics aimed at cancer-related signaling pathways.