Protocol for deriving proximity, affinity, and stoichiometry of protein interactions using image-based quantitative two-hybrid FRET

Two-hybrid Förster resonance energy transfer (FRET) provides proximity, affinity, and stoichiometry information in binding interactions. We present an image-based approach that surpasses traditional two-hybrid FRET assays in precision and robustness. We outline instrument setup and image acquisition and further describe steps for image preprocessing and two-hybrid FRET analysis using provided software to simplify the workflow. This protocol is compatible with confocal microscopes for high-precision and imaging plate readers for high-throughput applications. A plasmid-based reference system supports fast establishment of the protocol. For complete details on the use and execution of this protocol, please refer to Rivas et al.1.