Seminarium Zakładu Biofizyki

Among many existing fluorescence-based applications, dedicated to characterization of molecular interactions, vast majority depend on site-specific labeling, binding- induced change of conformation, or size of interacting molecules. To overcome these limitations, we applied a ratiometric dual-emission approach that quantifies ligand-induced spectral shifts with sub-nanometer sensitivity. The use of environment-sensitive near-infrared dyes with the method we describe, enables affinity measurements and thermodynamic characterization without the explicit need for site-specific labeling or ligand-induced conformation changes. The newest isothermal spectral shift technology, implemented together with TRIC (temperature related intensity change), in latest NanoTemper’s system, Monolith X allows researchers to work in solution with variety of biomolecules, including proteins, antibodies, and nucleic acids, as well as with the most challenging types of targets, like membrane, intrinsically disordered proteins, or PROTACs. The method is immobilization free, requires only a few microliters of sample per data point, and experiments can be done in any buffer, including crude lysate or serum.