ITC Technical Tip: Merging ITC data files by concatenation

Concatenation (noun) con·cat·e·na·tion plural concatenations

Definition of concatenation:

1. a group of things linked together or occurring together in a way that produces a particular result or effect
2. the act of concatenating things or the state of being concatenated : union in a linked series

(merriam-webster.com)

Isothermal Titration Calorimetry (ITC) is an analytical method used to determine binding affinity, binding enthalpy, and binding stoichiometry in a single titration experiment.  By the end of the experiment, all the binding sites of the “macromolecule” in the ITC cell should be occupied by the “ligand” added throughout the ITC experiment.  For most ITC experiments, binding saturation is achieved by starting with an appropriate concentration of the ligand in the ITC syringe, and introducing all of the syringe contents into the ITC cell via a series of injections. 

However, there are situations where the binding is  not saturated after one syringe-full of ligand had been added to the macromolecule in the ITC cell, and you need to add more ligand to achieve binding saturation.  This can occur when:

• The ligand concentration in the ITC cell is too low to saturate all the binding sites
• The macromolecule has multiple binding sites, and the ligand concentration in the ITC syringe is not sufficient to saturate all of the sites
• The binding affinity is weaker than expected, and a higher ligand concentration is needed to achieve binding

It is possible to perform the 2^nd series of injections (and more, if needed), and concatenate the resulting ITC data files for analysis as a single data file.

This can be done with any MicroCal ITC (PEAQ-ITC, iTC200, VP-ITC), and the automated PEAQ-ITC and automated iTC200 systems

1. Perform the 1^st ITC experiment using standard protocols
2. After the 1^st experiment is completed, do not remove material from the ITC sample cell, and do not clean the sample cell. 
3. Carefully remove any material that is in the overflow reservoir above the ITC cell’s filling stem (optional)
4. Refill the ITC syringe with the same ligand solution as used in the 1^st experiment
5. Perform another ITC experiment, using the same method as the 1^st experiment
6. If needed, repeat steps 2-5.

Note: If you use the automated MicroCal PEAQ-ITC or the automated MicroCal iTC200, be sure the automation method for the 2^nd (and subsequent) titrations is “Continue Injections” – this automation protocol will remove 40 microL from the overflow reservoir, skip the cell cleaning, and refill the syringe.  You will only need fresh ligand for these experiments.

For the concatenation, you need to download the MicroCal Concat software.

Follow the software instructions to import the 1^st and 2^nd experiments, and create a 3^rd filename for the concatenated data.  If you need to join 3 experiments, take the concatenated file for experiments 1 and 2 as the 1^st experiment, and add the 3^rd data file to that to create a new concatenated file.  You can continue to create and add data files if needed.

The resulting data file can be analyzed as a single experiment.

Here is an example of data concatenated from  4 experiments: