Isothermal titration calorimetry

MicroCal Auto-iTC200 specifications

• Operating temperature: 2 °C to 80 °C
• Sample storage temperature: 4°C to ambient
• Response time: 10 seconds
• Cell design: 200 µl coin-shaped
• Titration syringe: 40 µl
• Usable syringe volume: 38 µl
• Smallest injection size: 0.1 µl
• Stirring speed: 500-1,500 rpm

All samples and buffers should be filtered by 0.2 μm filter and degassed

Buffers

• Buffers for macromolecule (M) and ligand (L) must be identical to minimize ΔH from dilution.
• If both M and L are large, dialyze simultaneously into the same buffer using separate dialysis vessels.
• If L is too small to be dialyzed, use the “dialysate” to dissolve solid ligand.
• Reducing agents: DTT is not recommended; substitute with β-mercapoethanol or TCEP (not compatible with phosphate buffer) if possible.
• To minimize artifactual heats, the buffer should have a low enthalpy of ionization (e.g.phosphate, citrate, acetate).
• 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.
• A difference in [DMSO] between M and L will yield large ΔH from dilution and obscure binding data. If you use DMSO to solubilize a ligand, you will need to add DMSO to the macromolecule solution to match the concentration of 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 before running the ITC experiment.

Samples

• Sample volume (Auto-iTC200)
  • Sample cell (Macromolecule) → 430 μL per run;
  • Syringe (Ligand) → 150 μL per run
• Sample concentrations
  • Typical starting concentrations are 30-50 μM for the sample cell.
  • Generally, the syringe concentration is 10x higher than the concentration in the sample cell; 7x higher for very tight binding and 10-15x (or more) for very weak binding.