Protein Crystallography and
Biophysics Centre (BiophysX)
Institute of Structural and Molecular Biology
(ISMB)
Birkbeck College / University College London
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
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.
References:
Sultana and Lee, Current Protocols in
Protein Science, 79:19.25.1-19.25.26
(2015)
