Multicolor Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein Interactions with Alternative Partners

Many proteins can interact with several alternative partners. The multicolor bimolecular fluorescence complementation (BiFC) assay enables simultaneous visualization of multiple protein interactions in the same cell. This assay, introduced here, is based on the fusion of fragments of fluorescent proteins that form spectrally distinct BiFC complexes to different interaction partners. These complexes can thereby be spectrally resolved and the relative amounts of complexes formed with different interaction partners can be determined. The multicolor BiFC assay enables comparison of the subcellular distributions of complexes formed with different interaction partners and allows analysis of the competition between mutually exclusive interaction partners for binding a shared partner present at limiting concentration.