Protein Crystallography and Biophysics Centre (BiophysX)
Institute of Structural and Molecular Biology (ISMB)
Birkbeck College / University College London

Bio-Layer Interferometry

Bio-Layer Interferometry (BLI) is a label-free technology for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time.

The binding between a ligand immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift, Δλ, which is a direct measure of the change in thickness of the biological layer. Interactions are measured in real time, providing the ability to monitor binding specificity, rates of association and dissociation, or concentration, with precision and accuracy.
Only molecules binding to/or dissociating from the biosensor can shift the interference pattern and generate a response profile on the Octet® System. Unbound molecules, changes in the refractive index of the surrounding medium, or changes in flow rate do not affect the interference pattern. This is a unique characteristic of BLI and extends its capability to perform in crude samples used in applications for protein:protein binding, quantitation, affinity, and kinetics.

Sample requirements
  • Plan an entire day for the first experiment. Depending on optimisation 2-3 days are required to achive the desirable data quality

  • Biosensors and immobilization methods:
    Please see

  • Sample to be immobilized: A few μL at nM concentration is usually enough.
    The binding partner: The concentration required will depend on the KD to be measured, hence requires an initial experiment (see below: Titrations). The run at each concentration may still be done in 4-5 μl drops. However, if long incubation times are required, then 200 μl of each concentration are needed.
  • Controls: They are crucial for interpreting the results: 
    - Negative control (for unspecific binding): 
          Perform each run also on a biosensor with nothing immobilized on it.
    - Two immobilization levels (for mass transfer):
          Perform each run on two different immobilization levels.
          For kinetics this ensures that it is not dominated by diffusion limits.
          For affinity it ensures it is not influenced by steric hindrance.
    - buffer blanks (bulk signal):
         Perform a buffer blank on each used tip to check for signal offsets.
  • Titrations:
    Generally 10 concentrations are needed, 5 each above and below the KD.

Please contact us for detailed protocols and planning your experiment.

ISMB Protein Crystallography and Biophysics Centre, Birkbeck, University of London
Last modified April 2021