Bimolecular fluorescence complementation detects protein-protein proximity in living or fixed cells by fusing two candidate interaction partners to nonfluorescent N- and C-terminal fragments of a fluorescent protein; when the partners interact or remain close enough, the fluorescent fragments complement, mature, and generate a fluorescent signal at the site of the protein complex. The BiFC readout is fluorescence intensity and subcellular localization of the reconstituted fluorophore, which reflects formation or stabilization of a protein complex rather than direct biochemical binding kinetics; BiFC is therefore useful for mapping where interactions occur in cancer cells, neurons, macrophages, organoid-derived cells, or drug-screening systems, but results should be validated by independent assays such as co-IP or Western blot. BiFC signal formation is delayed by fluorophore maturation and can stabilize otherwise transient complexes, so it is not a real-time reversible interaction assay
Bimolecular Fluorescence Complementation (BiFC) Assay
Bimolecular Fluorescence Complementation (BiFC) is a biological imaging technology used to study protein interactions. By connecting two parts of the target protein to two complementary fragments of a fluorescent protein, when the two parts bind to each other, the fluorescent protein reassembles, producing a fluorescent signal. BiFC can monitor the spatiotemporal dynamics of protein interactions in living cells in real time, providing an intuitive, high-resolution tool for cell signaling and protein interaction research.
Related Experimental Schemes
