Isothermal Titration Calorimetry – FAQs
What kind of molecular interaction can I measure?
You can measure bi-molecular interactions between any kind of molecule: protein, DNA, RNA, small molecule compounds, lipids, carbohydrates
What are the required compound concentrations?
In general, the concentration of the ligand in the syringe should be 10-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 30-times KD. If KD is unknown, a best guess will do as well. The concentration of the interaction partner inside the syringe should be around 10*n-times that of the partner in the sample cell, where n is the stoichiometry of the reaction. The following concentrations are frequently used to determine KD values between 10 µM and 10 nM: The interaction partner that is present in the sample cell should be in the concentration range of 10 µM to 30 µM. The other interaction partner, which is titrated, should typically be in a concentration range approximately 10-fold higher, thus between 100 µM and 300 µM (under the assumption of a 1:1 binding stoichiometry). However, the necessary concentration range depends on the heat released or absorbed during the specific interaction.
How much sample do I need?
To fill the sample cell, 280 µL are needed. The minimum volume to fill the syringe is 40 µL, although we recommend that 60 µL be used to avoid bubbles or foaming during automatic loading of the syringe.
How high is the throughput?
Establishment of specific experimental conditions usually requires one working day. After that, depending on the number of injections and time spacing between injections, around 6-10 experiments are possible over a typical working day.
What is the temperature range?
10°C to 80°C
Can I measure without labeling molecules?
Yes, ITC does not require any labeling prior to the measurement.
Can I measure without immobilizing molecules?
Yes, ITC does not require any immobilization on a surface prior to the measurement.
What type of information do I get?
The ITC technology is not only able to determine binding affinity and interaction stoichiometry, but also to generate a complete thermodynamic profile of the interaction including the binding enthalpy ΔH, the binding entropy ΔS, as well as the free enthalpy of binding ΔG.
Which range of affinities can be quantified?
ITC can determine binding affinities in a wide range from nanomolar (10-9) to millimolar (10-3) affinity.
Can I measure binding kinetics with ITC?
ITC measures equilibrium binding constants and it is this not possible to determine association and dissociation rates (kon, koff)
Can I measure the binding of small molecules?
Yes, ITC is not only able to characterize the interaction between macromolecules like proteins or nucleic acids, but also between macromolecules and small molecular inhibitors, effectors, or the like.
Are there limitations to the buffers or additives I can use?
In general, the buffer or additive should not negatively affect the stability or homogeneity of the sample. Moreover, it is important to minimize artefactual heat by using a buffer with a low enthalpy of ionization (e.g. phosphate, citrate, or acetate buffers). Common biological buffers with amino groups such as TRIS or HEPES are not recommended for ITC due to their high heats of ionization. Also, avoid using DTT if possible, because DTT can cause high background heat. If a reducing agent is required nonetheless, use β-mercaptoethanol or TCEP at a maximum concentration of 1 mM.
Do I need to prepare my samples in a specific way?
It is important for a successful and high-quality ITC experiment that both interaction partners are in identical buffer solutions. Other large heats of dilution can mask the actual heat generated or absorbed by the interaction. Usually it is best practice to thoroughly dialyze both samples (interaction partners) against the same buffer prior to the ITC experiment or to use desalting columns for buffer exchange.
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.
Can I measure samples that contain DMSO?
Yes, this is possible. If one interaction partner (e.g. a small peptide dissolved in DMSO) contains DMSO, it has to be added to the solution of the second interaction partner (e.g. protein in the sample cell) to the same concentration in order to avoid buffer mismatch and high dilution heats. Usually 2-5 % of DMSO can be added to protein solutions in the short term.