Biolayer Interferometry – Comparison

MicroScale Thermophoresis - binding parameters with great precision…

Biolayer Interferometry – Comparison to other biophysical techniques

Next to BLI, important biophysical methods used to investigate molecular interactions include Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), and MicroScale Thermophoresis (MST). Certain advantages and drawbacks of these methods are listed below.

 

FACTS & FEATURES: BLI compared to other methods
MethodBiolayer Interferometry (BLI)
Affinity Range (Kd)
  • sub nM to mM
Advantages
  • Fast and easy to perform
  • Determination of binding kinetics possible (kon, koff)
  • Crude sample compatibility
  • Immune to fluctuations in refractive index of samples
  • No microfluidics involved (as with SPR)
  • Less expensive consumables (compared to SPR)
Draw Backs
  • Immobilization required
  • No complete thermodynamic profile of the interaction
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
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
MethodMicroScale Thermophoresis (MST)
Affinity Range (Kd)
  • pM to mM
Advantages
  • measurements in solution
  • free choice of reaction conditions
  • low material consumption
  • temperature controlled
  • fast measurements
  • interaction between small and big molecules detectable
  • fluorescence and labe-free assays
  • multiplex assay
Draw Backs
  • Kon and Koff rates are not accesible