Operation of forteBIO Molecular Interactions Instrument
The fortéBIO Red Octet 96 instrument uses BioLayer Interferometry to measure binding of molecues to a protein immobilized on a probe. Although the measuring the amount of bound material is done in a different way from surface plasmon resonance (SPR) in the well known Biacore instruments, most of the experimental design is similar.
There are several ways to attach a protein or other molecule to a probe. The available probes are at:
If the ligand has no tag, the company suggests using the streptavidin probe after biotinylating a protein with a biotinylation kit, aiming for one biotin per molecule. This attachment scheme avoids the complexity of determing a suitable pH as needed when using amine coupling. Amine buffers have to be removed for biotinylation. For a procedure, see:
Biotinylation of proteins (fortéBIO)
Application note 28
As with SPR, there is an optimal amount of protein to couple, which is less than the maximum achievable. Large quantities of protein on the probe may give inaccurate results because of mass transfer limitations. With the Octet, a shaking movement of the probes in a solution of analyte is the equivalent of the linear flow in an SPR instrument.
The Octet can read 8 probes simultaneously, so that measurements can be fast after finding suitable conditions. The streptavidin probes are relatively cheap and thus often used only once for kinetic measurements, saving the time and materials consumed by looking for good regeneration conditions as is common for SPR experiments. Small molecules often dissociate rapidly, so that after a relatively short wait, effectively all analyte has dissociated from the probes which can be used again.
At the end of the readings, the analyte solution can be removed from the plate and used for other purposes; the amount removed during binding is a small fraction of the total.
Requirements for use
- Reserve the instrument through iLab or collab.
- Purchase probes (96/tray) from fortéBIO (the core may have streptavidin probes available) and 96 well plates (Greiner all black polypropylene); suggested shelf life of probes is 6 months.
- Minimum sample volume of 180 µL; smaller volumes may cause signal artifacts
- Analyte concentration 5 pM to 1 mM, or if the range is known, 0.1 x KD to 10 x KD.
Biomolecular Binding Kinetics Assays on the Octet Platform
Application note 14
Application note 8