Biolayer Interferometry (BLI) to Quantify RALF1-Pectin Interactions.

Cellular function relies on a network of precisely regulated interactions among macromolecules such as proteins, peptides, carbohydrates, and nucleic acids. These molecular interactions regulate vital processes, including signaling, structural organization, and developmental patterning. Biolayer interferometry (BLI) is a label-free optical biosensing technique that enables real-time quantification of such interactions. This protocol describes how to use BLI to assess the binding affinity between a biotinylated plant peptide hormone (RALF1) and cell wall-derived oligogalacturonides (OG25-50) on the Octet RED96 platform. Streptavidin-coated biosensors are employed to immobilize the ligand, while analyte binding is monitored through wavelength shifts in the reflected light. The protocol includes detailed steps for sensor preparation, assay setup, software configuration, and kinetic data analysis. While optimized for plant peptide-matrix interactions, the method is broadly adaptable to other macromolecular systems across biological disciplines. Key features • This protocol requires optimizing ligand and analyte concentrations to achieve optimal results. • The buffer composition plays a vital role in supporting ligand-analyte binding, as their interaction is influenced by charge.