ITC Technical Tips: Sample Preparation

Sample preparation is a key step in achieving quality results with ITC.  Both ligand and macromolecule have to be in IDENTICAL solutions (pH, buffer composition, ionic strength, reducing agent, detergent, etc.).  ITC is very sensitive, and even small buffer mismatches can generate large heats of dilution with each injection, which can mask heat changes from ligand binding.

Choice of solution conditions

Use a buffer in which ligand and macromolecule are stable and soluble.  Be sure buffer concentration is high enough to compensate for any pH effects during titrations.  ITC can be performed with all biological buffers, at a wide pH range.  For a full characterization of binding, you can perform titrations at different pHs, buffer compositions, ionic strengths, etc.

Reducing agent

If you need a reducing agent, we suggest TCEP or beta-mercaptoethanol, rather than DTT.  Use of DTT can result in aberrant baselines.


Before ITC, dialyze ligand and macromolecule against the same dialysis buffer.  At the end of dialysis, save several milliliters of dialysis buffer to be used for dilution and control ITC experiments.

Note that some buffer components, like glycerol and detergents, dialyze slowly, so use of these additives requires longer dialysis time.

If one of your components is too small for dialysis, dialyze the larger component, and use the final dialysis buffer to solubilize the smaller component.

Concentration and pH

After sample preparation, check the concentration and pH of both ligand and macromolecule solutions.  Use these concentrations for data analysis.  If the pHs differ by more than 0.05 pH unit, titrate one solution with acid or base until both pHs are matched.

Synthetic peptides and oligonucleotides

Be sure they are desalted PRIOR to suspension in ITC buffer — residual chemicals from the synthesis (e.g. TFA and salts) will cause a buffer mismatch and high heats of dilution.

Organic solvent

If you need DMSO to solubilize a ligand, you will need the same DMSO concentration in the ligand and macromolecule solutions.  Many proteins are stable (in the short term) in up to 2-5% DMSO.  Check ligand solubility by serial dilutions of 100% DMSO stock solution in buffer.  Immediately before the ITC experiment, add DMSO to protein solution to match DMSO concentration in ligand solution.

From MicroCalorimetry News, MicroCal, LLC, Northampton, MA 01060