Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells

Nat Protoc. 2006;1(3):1278-86. doi: 10.1038/nprot.2006.201.


Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.

MeSH terms

  • Fluorescent Dyes / metabolism*
  • Genetic Complementation Test / methods*
  • Microscopy, Fluorescence
  • Multiprotein Complexes / analysis*
  • Multiprotein Complexes / metabolism
  • Spectrometry, Fluorescence / methods*


  • Fluorescent Dyes
  • Multiprotein Complexes