Isothermal Titration Calorimetry – Comparison to other biophysical techniques

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Isothermal Titration Calorimetry – Comparison to other biophysical techniques

Next to ITC, important biophysical methods used to investigate molecular interactions include Surface Plasmon Resonance (SPR), Biolayer Interferometry (BLI), and Size Exclusion Chromatography (SEC). Certain advantages and drawbacks of these methods are listed below.

 

FACTS & FEATURES: ITC compared to other methods
MethodIsothermal Titration Calorimetry (ITC)
Affinity Range (Kd)
  • nM to µM
Advantages
  • Label-free
  • In solution (no immobilization required)
  • Complete thermodynamic characterization (ΔH, ΔS, ΔG)
  • Determination of binding characteristics (KD, stoichiometry)
  • Wide range of possible buffers and solvents
  • No limitations in molecular weight
  • No expensive consumables
Draw Backs
  • Low throughput (6-10 experiments per working day)
  • Sample consumption can be too high in certain cases
  • Not possible to determine binding kinetics
  • Interactions with no or only a small change in enthalpy not measurable
MethodSurface Plasmon Resonance (SPR)
Affinity Range (Kd)
  • sub nM to low mM
Advantages
  • High sensitivity
  • Label-free
  • Determination of binding kinetics possible (kon, koff)
Draw Backs
  • Lab-intense establishment of new assays
  • Immobilization required
  • No complete thermodynamic profile of the interaction
  • Expensive consumables
  • Not possible in complex biological liquids
  • Artifacts stemming from mass transport limitations close to an interface
MethodBiolayer Interferometry (BLI)
Affinity Range (Kd)
  • sub nM to mM
Advantages
  • Label-free
  • Determination of binding kinetics possible (kon, koff)
  • Possible in complex biological liquids
  • No artifacts from mass transport limitations as in SPR
Draw Backs
  • Immobilization required
  • No complete thermodynamic profile of the interaction
MethodSize Exclusion Chromatography (SEC)
Affinity Range (Kd)
  • µM to mM
Advantages
  • Fast and easy to perform
Draw Backs
  • In most cases only possible for biomolecules with UV-absorbance or fluorescence
  • Determination of binding constant and thermodynamic parameters of an interaction not possible
  • Relatively high sample consumption (dependent on detection method)