Bimolecular fluorescent complementation (BiFC) assay for visualization and characterization of protein –protein interactions in living cells

Finding a trustworthy method to recognize protein-protein interactions is going to be a big task in the future. Bimolecular fluorescent complementation (BiFC) assay 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 interact and form a fluorescent complex. Protein function is often mediated by formation of stable or transient complexes. The BiFC assay involves splitting of yellow fluorescent protein into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in a frame to a gene of interest, enabling expression of fusion proteins. No fluorescence was detected following co-expression free non-fused YN and YC or non-interacting protein pairs. Reconstitution of a fluorescing yellow fluorescent protein (YFP) chromospheres occurred only when the inquest proteins interacted. It can be used in visualizing the interaction of a phytoplasma effector with its proteinaceous host partner in Nicotiana benthamiana mesophyll protoplasts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different sub cellular locations and in different cell types and organisms. This assay growing popularity is a result of its simplicity, usability, and capacity to conduct experiments. It is technically straight forward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents