- Bimolecular Fluorescence Complementation (BiFC) is a powerful technique used to study protein–protein interactions in living cells by exploiting the reconstitution of a fluorescent protein from two non-fluorescent fragments.
- The method is based on the principle that a fluorescent protein, such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP), can be split into two non-functional halves. When each half is fused to a protein of interest, the interaction between these two proteins brings the fragments into close proximity, allowing them to reassemble into a functional fluorescent protein. The restored fluorescence serves as a direct and visible readout of protein–protein interaction.
- In a typical BiFC experiment, two proteins hypothesized to interact are genetically fused to the N-terminal and C-terminal fragments of a split fluorophore. These fusion constructs are then co-expressed in living cells. If the target proteins physically interact, the fluorophore halves come together and fold into a fluorescent complex, emitting detectable fluorescence that can be visualized using standard fluorescence microscopy. This fluorescence is often stable and spatially localized, allowing researchers to observe not only whether an interaction occurs, but also where within the cell the interaction takes place.
- One of the main advantages of BiFC is its simplicity and ability to directly visualize protein interactions in the native cellular environment, without the need for external substrates or cofactors. It allows detection of relatively weak or transient interactions that may be difficult to capture using biochemical techniques. Moreover, BiFC is highly specific, as reconstitution of fluorescence typically occurs only when the tagged proteins are in very close physical proximity (within 5–10 nm), reducing the likelihood of false positives due to random proximity.
- However, BiFC also has limitations. The complementation of the fluorescent protein is generally irreversible, which means that dynamic interactions may be trapped and cannot be temporally resolved once the fluorophore is reassembled. This irreversibility can lead to overestimation of interaction strength or persistence. Additionally, the reconstitution process may sterically hinder protein function or interfere with proper folding, necessitating careful controls and optimization of fusion protein design.
- In summary, BiFC is a valuable technique for detecting and visualizing protein–protein interactions in live cells with high spatial resolution. While it is not suitable for studying rapid or reversible interactions, its robustness and simplicity make it an excellent choice for qualitative analysis of protein complex formation and subcellular localization of interactions in situ.
