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ITC Notes

Background Information

Isothermal Titration Calorimeter (ITC) is most useful for measuring dissociation constants in the 10-5 to 10-8 M range. For very tight binding (nM), the optimal concentration of macromolecule needed is so low that the heat changes upon binding become too small to measure. Conversely, weak binding (~100uM) requires very high concentrations and heat changes can be obscured by aggregation and non-specific binding.

How to use the Affinity ITC

Follow these guidelines for proper sample preparation, which is critical to achieve good ITC data.

Buffers

  • It is important to use a buffer and pH conditions in which the proteins are stable.
  • To minimize artifactual heats, the buffer should have a low enthalpy of ionization (e.g. phosphate, citrate, acetate). Quaternary amines (e.g. Tris) has high enthalpy of ionization. For information, refer to:

Goldberg, R.N., Kishore, N., and Lennen, R.M. Thermodynamic Quantities for the Ionization Reactions of Buffers. J. Phys. Chem. Ref. Data, Vol. 31 No. 2.

  • Be sure your buffer concentration is high enough to compensate for any pH effects during titrations.
  • Avoid using DTT as it can cause erratic baselines. If you must include a reducing agent, use β-mercaptoethanol (BME) or tris(2-carboxyl) phosphine (TCEP). There are literature reports where people use up to 5mM BME without any problems but lower concentrations (1 mM) are recommended, especially if your heats of binding are small. TCEP is not stable in phosphate buffer.

Typical Volumes

  • In a typical experiment, 300 µL of deionized H2O and protein sample are loaded into the reference and sample cells, respectively. Do not add buffer into the reference cell.
  • The injection syringe has a volume capacity up to 250 µL. However, the Manual Syringe Load Wizard will show the volume needed to be load into the injection syringe based on the volume of injection and the total number of injections selected in the program.
  • Keep in mind that you may need sample for controls and test runs, so more is better.
  • The following review provides practical information on planning ITC experiments:

Freyer, M.W. and Lewis, E.A. Isothermal Titration Calorimetry: Experimental Design, Data Analysis, and Probing Macromolecules/Ligand Binding and Kinetic Interactions. Methods in Cell Biology. 2008. 84:79-113.

Sample Preparation

  • Both macromolecule and ligand must be in identical solutions, otherwise large heats of dilution will mask the desired observation. Solvent matching is best achieved by exhaustive dialysis of the macromolecule, using the final dialysis buffer to make up the ligand solution. If both components are macromolecules, they may be dialyzed in the same pot.
  • Save some dialysis buffer (>20 mL) for rinsing cells, running baseline controls, diluting samples, etc.
  • All the samples, including the deionized H2O and the buffer used in the experiment, need to be filtered and then degassed for at least 10 minutes before their use.
  • After the solutions have been prepared, check their pH carefully. If they differ by more than 0.05 pH units, then adjust one of the solutions so they match.
  • Solution concentrations should be determined after final preparation. Accurate determination of binding parameters is only possible if concentrations are known precisely.
  • The accuracy of stoichiometry, dissociation constant and enthalpy (N, Kd and ΔH) determination is directly proportional to the accuracy with which the syringe reactant concentration is determined. This contrasts with the accuracy of the cell reactant concentration which only affects N.
  • If you are working with synthetic peptides or oligonucleotides, be sure they are desalted prior to suspension in ITC buffer. Residual chemicals from synthesis (e.g. TFA and salts) will cause a buffer mismatch and high heats of dilution.
  • If you use DMSO to solubilize a ligand, you will need to add DMSO to the macromolecule solution to match the concentration in the ligand solution. Many proteins are stable in the short term in up to 2-5% DMSO. Add the DMSO to the protein solution immediately prior to running the ITC experiment.

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