Isothermal titration calorimetry

What is ITC analysis used for?

Isothermal titration calorimetry is most commonly used in biochemical studies to analyze the interactions between larger molecules, such as proteins, enzymes, and DNA, with small ligand molecules. The technique can measure factors such as stoichiometry, binding constant, enthalpy, and entropy changes associated with biomolecular interactions. One of the most common ITC applications is drug development, as the technique provides information on how effectively medicinal compounds bind to target molecules within the body.

How does isothermal titration calorimetry work?

Whenever a chemical interaction between two species occurs, there will be a very small change in the temperature of the surrounding solution as energy is either absorbed or released. By measuring the temperature changes it is possible to determine the exact nature of the interaction.

ITC works off of this principle by measuring the power it takes to keep the temperatures of two cells constant, one as a reference and one containing the biomolecules of interest. A solution containing the ligand molecule to be tested is injected into the test cell and begins to interact with the main biomolecule inside. The calorimeter monitors the power needed to reset the minuscule changes in the temperature of the test cell. By repeating this process and logging temperature changes over time, information is obtained on the thermodynamic properties of the interaction

Sample requirements and preparation

ITC analysis is performed in solution, so both the larger biomolecule and the test ligand will need to be in a suitable concentration for the test to be performed. Due to the nature of the types of molecules that are usually tested, a buffer solution may be implemented to ensure that pH is kept within a certain range throughout testing. In this case, both the biomolecule and ligand samples must be incorporated into the same buffer to minimize interference.

Advantages and limitations of ITC analysis

The key advantage of ITC is that it can be used to monitor biochemical interactions in a non-destructive way. It does not require the use of chemical markers or other preparation steps that could adversely affect the samples. It can monitor chemical interactions in solution, providing a suitable model for how interactions occur inside the body. Finally, ITC is non-specific, meaning that it can be used to monitor a very wide range of biological interactions, regardless of the exact chemicals involved in them.

The downside to ITC is that the signal provided during chemical interactions can be minuscule, to the point that in some cases it is difficult to detect. Therefore, a relatively large sample is often required to obtain substantial results; more so than in most other biochemical assays. To achieve the best results, each sample must be tested over a relatively long period, leading to a low general throughput of samples. Furthermore, some samples can be susceptible to contamination or changes in pH, so careful sample preparation is required.

ITC vs. DSC – what are the differences?

Differential scanning calorimetry (DSC) is a thermal analysis technique that is used to measure changes in a sample across a temperature range. Here, the sample is exposed to a temperature ramp. The energy required to change the temperature of the sample is recorded, helping to reveal how the sample absorbs energy compared to the reference.

The key difference is that in DSC the sample is exposed to a range of external temperatures, making it more suitable for measuring how processes will change under these conditions. This makes it ideal for studying the stability of proteins and other molecules under adverse conditions. ITC, on the other hand, is used at a set temperature and is more focused on monitoring the chemical processes themselves, rather than the effect of external conditions.

Need ITC analyses?

Measurlabs offers laboratory testing with ITC methods for various analytical purposes. From tens to hundreds of samples, we ensure your analyses are handled on time, with the highest quality. Our experts are just a message away, ready to guide you from method selection to result interpretation with clarity and care. Reach out to us using the form below to request a quote and we will get back to you latest the next business day.