ITC sample preparation
Sample preparation is critical for achieving good ITC data, so below are some guidelines. For more information on ITC and its applications, check out the Malvern website.
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.
- To minimize artifactual heats, the buffer should have a low enthalpy of ionization (e.g. phosphate, citrate, acetate). Refer to: J. Phys. Chem. Ref. Data (2002) 31:2 p.359.
- 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. This is something you'll need to work out for your system.
- The volume of the macromolecule solution (in the reaction cell) should be ≥2.1 mL per titration. The ligand solution (in the injection syringe) should be ≥0.7 mL per titration. These volumes exceed the capacity of cell/syringe, but are necessary in order to fill them easily.
- Keep in mind that you may need sample for controls and test runs, so more is better.
- Other considerations for designing ITC experiments (concentrations, temperature, run parameters, etc.) can be downloaded here as a pdf document.
- This review in Methods in Cell Biology, Vol. 84 (2008) provides a lot of practical information on planning ITC experiments.
- 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 dialysed in the same pot.
- Save some dialysis buffer (>20 mL) for rinsing cells, running baseline controls, diluting samples, etc.
- Filter out any visible particulates.
- 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, association constant and enthalpy (N, Ka and ΔH) determination is directly proportional to the accuracy with which the syringe reactant concentration is determined. This is in contrast to 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.