The quantity of heat measured is in direct proportion to the amount of binding. In the example below, the reaction is exothermic, which means the sample cell becomes warmer than the reference cell and causes a downward peak in the signal. As the temperature of the two cells comes back to being equal, the signal returns to its starting position.
The second small aliquot of the ligand is injected into the sample cell and once again the microcalorimeter compensates for the small heat changed detected. The molar ratio between the ligand and protein is gradually increased through a series of ligand injections. The protein gets more and more saturated, less binding of the ligand occurs and the heat change starts to decrease until ultimately the sample cell contains an excess of ligand versus protein, bringing the reaction towards saturation.
The area of each peak is then integrated and plotted versus the molar ratio of ligand to protein. The resulting isotherm can be fitted to a binding model from which the affinity K D is derived.
The molar ratio at the center of the binding isotherm give us the reaction stoichiometry. The plot shown below is an example of a binding reaction. This means that a single ITC experiment delivers a wealth of information about the binding reaction which helps understand the nature of the interaction and explore the thermodynamic drivers. Products Products. Product range. Product category. Measurement type. Industry standards. For the measurement exothermic and endothermic reactions are equally well suited.
Small values of deltaH, however, can make the reaction unmeasurable although the binding might be tight if the reaction is purely driven by the entropy deltaS. In order to test whether a reaction is not happening or just not measurable, one can try to change the value of deltaH into either direction by changing: a. Otherwise there will be large peaks due to the mixing and dilution of the buffers, especially if they have a different pH.
We found it useful to either dialyze or gel-filter both binding partners in the same buffer and also to keep some of that same buffer frozen for later repetitions. For ligands which may contain a high concentration of acid or base, such as lyophilized peptides from chemical synthesis, a high concentration of the buffering agent should be used to keep the pH constant when dissolving the ligand.
High noise in the experiment can also be caused by a bent injection syringe. Replacing the syringe is the best solution for this problem. Increasing noise during an experiment can be a sign of protein precipitation. Rinse the injection syringe with distilled water. Follow by a buffer rinse. Make sure the injection syringe is completely evacuated by drawing air through the system.
Place the needle of the injection syringe into the ligand solution and draw the ligand solution into the injection syringe until the entire syringe is full. Immediately close the fill port of the syringe and detach the tubing and plastic syringe. Purge and refill the injection syringe two more times to remove any bubbles from the syringe. Remove the syringe from the ligand solution and wipe the side with a kimwipe to remove any drops, being careful not to touch the syringe tip to the kimwipe as this may remove volume from the syringe.
Also, be careful not to knock or jar the syringe as this also can cause loss of volume from the syringe tip. Place the injection syringe into the sample cell. Set up the parameters for running the ITC. For binding systems with strong heat signals, a large number of low volume injections will give more data points for fitting e.
For systems that have weak heat signals, a small number of large volume injections are preferable e. Most commonly, fewer injections with higher injection volumes are used. It may take several titrations to optimize the conditions that are best for your system. It is important to note that the binding enthalpy can either be exothermic or endothermic, depending upon the system being studied. Unfortunately, some systems have low heat signals, making the heat of reaction difficult to determine.
Also consider the time spacing between each injection. It is imperative that after each injection of ligand, the system is given time to equilibrate and the heat signal returns to baseline before the next injection occurs. For most systems, three to five minutes should be adequate. The time between injections should be increased for systems where the equilibration doesn't occur within five minutes.
Because there will be some mixing between the macromolecule and ligand solutions in the injection syringe once it is inserted into the sample cell, the first injection will give spurious results. It is best to use small volumes e. It is best to choose a temperature that matches other experiments binding, kinetics, etc. The ITC can be set up to equilibrate at a temperature different from the temperature of the experiment.
This will decrease the time the instrument will take to reach the temperature of the experiment. The stirring speed of the syringe also needs to be considered. Stirring is necessary for adequate mixing of the ligand and macromolecule during the titration, but some proteins are destabilized by rapid stirring. For these cases, the stirring speed should be set at a relatively low rate. Once all the experimental parameters have been set up, then the experiment can be started.
Once the experiment has finished, the ITC can be cleaned according to the manufacturer's protocol. The solution in the sample cell at the end of the experiment can be kept if additional experiments on the macromolecule-ligand complex mixture are desired.
Repeat the titrations at least one or two more times to get reproducible data. Run a control where ligand is titrated into buffer in the sample cell to determine the heat of dilution for the ligand. For some systems where there is cooperative binding, additional information about the binding process can be gained by injecting the macromolecule into the ligand. The different injection orientations can give additional information, which may be helpful for global fitting.
Fitting of the data can be easily performed using macros in any data fitting program usually supplied by the manufacturer along with the instrument. Load the first data file. Check the raw thermogram for any signs of air bubbles or other artifacts in the signal.
If there are any artifacts such as spikes in the baseline or peaks when there was no injection at that time point , then note these data points as they should be removed. FAQ — General What kind of molecular interaction can be measured? With ITC it is possible to measure bi-molecular interactions between almost any kind of molecule, most importantly proteins, DNA, RNA, small molecule compounds, lipids, carbohydrates.
Can I measure without labeling molecules? ITC does not require any labeling prior to the measurement as it is a label-free method. Can I measure without immobilizing molecules? ITC does not require any immobilization on a surface prior to the measurement and the measurement takes place in solution. What information do I get from an ITC experiment?
Are thermodynamics possible? Additionally, ITC gives the stoichiometry of a molecular interactions and can also tell its affinity. Which range of affinities can be quantified? ITC can determine binding affinities in a wide range from nanomolar to millimolar affinity. Keep in mind that the weaker the affinity of an interaction, the higher the required samples concentrations are in order to yield a sufficient enthalpy signal.
Can I measure binding kinetics with ITC? ITC measures the thermodynamics and equilibrium binding constants of molecular interactions and it is thus not possible to determine association and dissociation rates binding kinetics. For the analysis of binding kinetics, please see Biolayer Interferometry. What is necessary for a successful determination of the thermodynamic parameters of an interaction? It is necessary to accurately determine the concentration of the interaction partner in the syringe.
Thus, it must be possible to precisely determine its concentration via spectroscopic means or color-based techniques. FAQ — Samples What are the required sample concentrations? In general, the concentration of the ligand in the syringe should be fold that of the interaction partner in the sample cell. In general, the concentration of the interaction partner inside the sample cell should be around times K D. If K D is unknown, a best guess will do as well.
However, the necessary concentration range depends on the heat released or absorbed during the specific interaction.
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