Biolayer interferometry is a method for measuring the affinity and kinetics of macromolecular interactions, involving proteins, nucleic acids, oligosaccharides, lipids… but also in some cases small ligands. One of the interaction partners has to be attached, either covalently or non-covalently, to the surface of a glass sensor, which is then dipped into a microplate well containing the other interaction partner. The association and dissociation between the interaction partners are monitored in real time at the surface, by following the variations of the optical depth of the surface layer by interferometry.
What are the sample requirements?
- Crude samples (such as culture supernatants or cell lysates) can be readily used to detect interaction and for comparison
- Sample concentrations are typically 1 and 50 µg/ml and volumes of 100-200 µl per sensor for the immobilized component.
- For interaction measurement, 100-200 µl of the interaction partners in solution are required at concentrations ranging from 1/10 to 10x the Kd. If the Kd range is unknown, optimization experiments need to be performed first to estimate the useful concentration range, before embarking into more precise studies to determine accurately the Kd and the association (kon) and dissociation (koff) rates.
- As for any biomolecular interaction analysis technique, optimisation and negative controls will require additional sample
What other specific considerations are relevant?
- The attachment of one of the macromolecules to a functionalized surface is required. A variety of immobilization procedures can be considered: covalent (via amide bonds) or non-covalent (via His6, GST or Fc tags, biotin moieties, …). BLI instruments are non-microfluidic systems which do not clog and are therefore well suited for the study interactions in crude samples such are cell lysates or culture supernatants.
- Octet instruments have multiple sensors, can measure 8 to 96 interactions simultaneously, depending on the model used, and as such are well suited for medium-scale screening studies.
Partners offering this technique
MOSBRI reference partner site for this technique: