Bimolecular fluorescence complementation relies on the facilitated complementation of splitted fragments of a fluorescent protein. By themselves these fragments don’t have any fluorescent properties but brought to close proximity they gain back the fluorescence. If BiFC works, it is a great way to show the dimeric proteins in live cells with a great spatial resolution. However, as in all other “too good to be true” stuff in life, BiFC suffers from some limitations. First, it really lacks a good temporal resolution due to the nature. When those fragments are brought together they need to complement, connect, refold bla bla bla 🥱and then gain back the fluorescence. Second crucial drawback is the formation of a “covalent bond”. It is believed that a bond is formed between the fragments in a way gluing the protein of interests together, unfortunately not an ideally “dynamic” way to image live cellular events.


Bimolecular luminescence complementation is pretty much similar with the BiFC, other than using splitted luciferases rather than splitted fluorescent proteins. It is known that 314 amino acid long Renilla luciferase (RLuc) can be splitted from position 229 and when those two RLuc fragments are brought to close proximity, they will refold and gain back the luminescence properties. Recently, NanoLuc was successfully splitted into two fragments from position 11, giving us the chance to use only a 11 amino acid fragment for tagging proteins. Furthermore, the complementation of NanoLuc is believed to occur reversibly, suggesting that the formation of a covalent bond is not necessary for regaining the luminescence properties. The kinetics of refolding is believed to be fast, however to the best of our current knowledge, we have no idea how fast it is.