Biolayer Interferometry – Technology
The Octet® K2 platform is a well-established tool for the kinetic analysis and characterization of molecular interactions between proteins, nucleic acids, and small-molecules. It is also widely used for quantification experiments as well as in drug discovery for kinetic screenings and kinetic optimization.
he Octet® platform is based on the optical and label-free analytical technique called biolayer interferometry (BLI). BLI analyzes interference patterns of white light that is reflected from two optical layers of a very small (600 µM diameter) tip: One internal reference layer inside the tip and one layer at the interface between the tip and the surrounding liquid (Figure below, panel A). Each reflection generates constructive and destructive interferences that vary with the wavelength (Figure below, panel B, gray curve). Any change at the outer layer of the tip (a biocompatible surface with one interaction partner immobilized on it), for example due to binding of a ligand, leads to different interference patterns at this reflective layer. This, in turn, causes a shift of the interference spectrum to different wavelengths (Figure below, panel B, red curve). From the time-resolved monitoring of this shift, it is possible to derive real-time association and dissociation rates of the ligands in solution to the immobilized interaction partner at the tip surface.
BLI allows for the real-time determination of the interaction dissociation constant (KD), as well as the observed association (kon) and dissociation (koff) rate constants. A wide range of different sensor tip surfaces allows the precisely tailored immobilization of one interaction partner. Common techniques for immobilization are direct surface immobilization using amine-reactive coupling, biotin-streptavidin based coupling, anti-GST– or anti-histidine-tag based coupling, and antibody-based coupling. Thus, BLI using the Octet® system generates a complete kinetic profile of a molecular interaction and by that, for example, help with a more-fine grained analysis of molecular interactions with similar equilibrium affinities.
Here at 2bind, the state-of-the-art the PALL FortéBio Octet® K2 System is used. This BLI system features two parallel measurement channels, a 96-well sample plate format, excellent sensitivity down to analytes of 150 Da, and high reproducibility. A sample consumption of only 200 µL and the possibility to re-use a sample for multiple measurements enables that analysis of precious and difficult to obtain analytes. The Octet® K2 system features a dynamic range of association and dissociation rate constants of six orders of magnitude (kon: 101 – 107 M-1s-1, koff: 10-6 – 10-1 s-1). Affinities (KD) can be determined in the range from 10-3 – 10-11 M. The lower and upper boundaries for quantification are 0.05 µg/mL and 2000 µg/mL, respectively.