Affinity Measurements

The Octet R8e is a BLI (Bio-Layer Interferometry)-based instrument used to measure the binding affinity between two molecules.

Samples are loaded into 96- or 384-well plates. The ligand is immobilized on an optical sensor, allowing up to eight samples to be analyzed simultaneously. The sensor is then incubated with the analyte, and the interaction is monitored in real time to determine the association rate (Ka), dissociation rate (Kd), and equilibrium dissociation constant (KD).

A wide variety of biosensors is available for capturing His-tagged, biotin-tagged, and Fc-tagged proteins. Alternatively, ligands can be immobilized using standard amine coupling chemistry. Specialized biosensors for nanobody capture are also available.

The Monolith NT.115 measures affinity in steady state by using small amounts of samples (ul range) as follows:
• One molecule (that is labeled with a fluorescent blue\green excited dye) is incubated with dilutions of the other molecules.
• The mixed samples are transferred to capillaries and subjected to a temperature gradient.
• Changes in fluorescence (due to heat or migration) are monitored
• A plot of change in fluorescence vs. concentration helps calculate the affinity at steady state.
The whole process can take less than 30 minutes.