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Isothermal Titration Calorimeter
(ITC)

image of ITC

The MicroCal VP-ITC is useful for the characterization of the thermodynamics of a binding reaction in solution.  In an ITC experiment, aliquots of a titrant (typically a protein, peptide or small molecule) are injected into the cell containing a macromolecule solution.  With each titration injection, the molecules interact and heat is either generated or absorbed.  The VP-ITC measures this heat of binding to determine the binding constants (K), reaction stoichiometry (n), enthalpy (ΔH) and entropy (ΔS). In addition, varying the temperature of the experiment allows the determination of the heat capacity (ΔCp) for the reaction.

Since the heat of binding is a naturally occurring event, the ITC does not require immobilization and/or modification of the reactants.  There are no limits on the protein or ligand size nor is the system dependent on the optical properties of the samples.  The major limitation of the ITC is that it requires relatively high concentrations of samples. 

Applications

  • To monitor binding interactions, such as but not limited to: antigen-antibody, DNA-drug, receptor-target, protein-ligand or protein-protein
  • Determination of reaction stoichiometry
  • Measurement of binding constants
  • To measure the thermodynamic properties of binding - enthalpy, entropy and Gibbs free energy

Specifications

  • Can accurately determine binding constants between ~102– 109 M-1
  • Operating temperature: 2oC to 80oC
  • Reference (water) cell volume: ~1.8 ml
  • Sample cell volume: ~1.5 ml
  • Titration syringe: total volume of 250ml; injection volumes typically 2 - 10ml
  • Strong acids are not to be used in the ITC

Sample Requirements

  • The buffer the ligand and macromolecule are dissolved in must be an EXACT match (in buffer composition, salt concentration, pH).  The pH should not differ by more than 0.05 pH units.
  • All cell and syringe solutions must be degassed before loading into the ITC
  • Reducing agents should be avoided due to their instability over time.  If reducing agents must be used, DTT should be replaced by b-mercaptoethanol or TCEP at concentrations less than 5 mM.
  • Typical ligand concentrations required are 10 - 15 times the macromolecule concentration; have at least 700ml available for a complete experiment. A typical ITC experiment consists of 2 titrations: titrating into buffer alone as well as into the macromolecule.
  • Typical macromolecule concentrations required are 5mM - 1mM; have at least 2.5 mls available for a single experiment

Supplies You Should Bring for Your Experiment

  • 3 ml syringes (2 per experiment)
  • At least 5mls of your buffer, degassed
  • If you plan to degas your solutions here using the ThermoVac, bring small stir bars
  • A floppy or ZIP disc to save your data

Training, Assistance or Instrument Problems

If you need training or some assistance with your ITC experiment, please contact Ben Ramirez PRIOR to scheduling instrument time.

If there is a problem with the instrument, please let Ben Ramirez know.  It is the responsibility of the previous user to clean the instrument.  If it is not clean when you arrive to use the instrument, please note it in the log book and tell Ben.

Manual and Articles

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