Isothermal Titration Calorimetry (ITC)
CMI ITC resources and user guide ⬇︎
Isothermal Titration Calorimetry (ITC) is a label-free method for measuring binding of any two molecules that release or absorb heat upon binding. ITC can be used to measure the thermodynamic parameters of biomolecular interactions, including affinity (KA), enthalpy (ΔH), entropy (ΔS), and stoichiometry (n). Energetically favorable binding reactions have negative free energy values, ΔG = RTlnKD. ΔG, has two energetic components, enthalpy (ΔH) and entropy (ΔS) and their contributions are expressed as: ΔG = ΔH -TΔS. In an ITC experiment, ΔH of binding is measured directly.
The microcalorimeter has two cells: one contains water and acts as a reference cell, the other contains the sample, into which a binding partner is titrated using an injection syringe. Heat sensing devices detect temperature differences between the cells when binding occurs in the sample cell and give feedback to the heaters, which compensate for this difference and return the cells to equal temperature.
The CMI has a Microcal ITC200 from Malvern.
Applications
- Equilibrium binding: KA, KD
- Stoichiometry of binding: n
- Macromolecular and small molecule binding
Key Features
- Truly modification-free (no labels or immobilization)
- No size limits
- Can gain insights into the mechanism of binding
- No consumables to purchase
ITC Resources
CMI ITC200 Getting Started Guide, general guidelines for sample preparation and standard protocol
MicroCal ITC200 Getting Started Handbook, Malvern Introduction to the ITC200
MicroCal ITC200 User Manual, Malvern instrument manual
Sample Preparation
Assay Buffers
- The two binding partners must be in identical buffers to minimize heats of dilution which can mask heats of binding.
- Additional buffer (matched to samples) is required for baselines, dilutions and rinsing the cell.
- DMSO has high heats of dilution and should be matched extremely well between the cell and the syringe.
- Small differences in pH will cause high heats of dilution.
- Reducing agents can cause erratic baseline drift and artifacts. TCEP is recommended over βMe and DTT. Avoid or keep ≤ 1 mM, especially if ΔH is small.
- Using degassed buffers will reduce the introduction of air bubbles and improve results.
Samples
- Required volumes for one experiment:
- ≥ 300 µL protein for sample cell (202 µL + ~ 80-90 µL for filling).
- ≥ 100-120 µL ligand for syringe (40 µl syringe + 20 µl for filling, for each injection)
- typical starting concentrations:
- 5-50 µM in the cell (at least 10x Kd)
- 50-500 µM in the syringe (≥10x concentration in cell for 1:1 stoichiometry)
- Aim for a c value between 10-100 for optimal fit
- Molar concentration should be accurately measured.
- Errors in Cell concentration affect stoichiometry.
- Errors in the Syringe concentration will directly translate to errors in the KD, and affect ΔH and n.
- Protein aggregates will interfere with ITC.
- Centrifuge or filter samples before use.
- Assess protein heterogeneity via light scattering.
- Purify protein samples with soluble aggregates by size-exclusion chromatography.
Supplies
Supplies provided by the CMI:
- 0.2 ml tubes for syringe filling
- water and methanol for instrument cleaning
C-Value
An important equation to consider when planning and optimizing an ITC experiment is the c value.
c = n•[M]cell/KD
n = stoichiometry
[M]cell=concentration in the cell
KD = dissociation constant
The c values determines the shape of your data. Ideally, the c value is between 10-100. Values of c that are too low (<10) can sometimes be used to fit KD, but can't be used to accurately deterimine stoichiometry. Values of c > 1000 can be used to accurately determine n, but not KD.
Concentrations should be chosen such that sufficient heat is produced (typically > 5 μM for protein in the cell) and such that c value is in the ideal range.