Bimolecular fluorescence complementation (also known as BiFC) is a technology typically used to validate protein interactions and interaction between proteins and other macromolecules that are essential for survival of cells. It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. This fluorescent signal can be detected and located within the cell using an inverted fluorescence microscope that allows imaging of fluorescence in cells. In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. Therefore, through the visualization and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest.
Principle of BiFC
The working principle of BiFC is based on the development of a fluorescent complex, as a result of the association of two segments of a fluorescent protein when they are in close proximity due to protein–protein interaction in the fragments, i.e., in BiFC, a fluorophore is divided into amino and carboxyl terminal ends. These ends are combined into two proteins. When these two proteins interact, both the segments re-associate resulting in re-formation of the fluorophore and fluorescence at the sites of interface.
Set of rules to design BiFC
The BiFC experiments as it is based on the interaction between the fluorescent protein segments takes place only under certain circumstances. BiFC analysis involves an assay that is fabricated in such a way that it satisfies all the parameters affecting the association of the fluorescent protein segments. The protocol of assay fabrication includes:
BiFC calibration is carried out in four stages as follows:
- Selection of appropriate fluorophore that works finely as BiFC synthesis partners. Examples of fluorophores are Venus and yellow fluorescent protein (YFP).
- Labeling of proteins in which the BiFC segments are bonded to the amino- or carboxyl-terminals of the selected protein.
- Determination of transfection circumstances before the testing of multiple mutants.
- Determination of changes that occur in the localization of proteins due to addition of BiFC segments.
Plating and cell transfection
In this process, the cells are initially plated in a glass bottom plate and in two wells of a 6-well plate and then they are allowed to settle overnight at room temperature. Then the cell DNAs are prepared for the transfection process. After completion of the transfection process, the combination is divided equally and then incubated at room temperature for a few hours. Then the transfection media is removed and incubated once again at room condition.
Preparation of cells for imaging
In this process, the cells are initially analyzed using an epifluorescent microscope in order to check their fluorescence. Then, the preparation of cells is done by addition of paraformaldehyde after which the cells are lysed to obtain the image.
Imaging of cells
The intensity of fluorescence is determined for every single cell. Selective analysis of BiFC signals is achieved by performing transfection along with CFP. Imaging is done using a confocal microscope.
Measurement of the intensity of fluorescence is accomplished using several imaging software.
Analysis of data
The efficiencies of fluorescence complementation are determined by the value obtained by division of the intensities of fluorescence complementation and the intensities of whole fluorescent protein in each cell.