Isothermal titration calorimetry for measuring macromolecule-ligand affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, c...

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Bibliographic Details
Published in:Journal of visualized experiments 2011-09 (55)
Main Authors: Duff, Jr, Michael R, Grubbs, Jordan, Howell, Elizabeth E
Format: Article
Language:English
Subjects:
Calorimetry - methods
Kinetics
Ligands
Macromolecular Substances - chemistry
Molecular Biology
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Summary:Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.
ISSN:1940-087X
1940-087X
DOI:10.3791/2796