Bimolecular fluorescence complementation

The bimolecular fluorescence complementation (English bimolecular fluorescence complementation, BiFC ) is a procedure in molecular biology for the detection of protein - protein interactions. The method is based on the complementation of two non-fluorescent fragments of a fluorescent protein such as green fluorescent protein (GFP). Superimposition of the two fragments of intact fluorescent complex is generated. The process of bimolecular Fluoreszkomplementation was largely developed by Tom Kerppola and employees.

Principle

The principle of the bimolecular fluorescence complementation is related to enzyme fragment complementation and two-hybrid systems. For the investigation of protein -protein interactions, two different fragments of a fluorescent protein are added to the two proteins to be investigated using molecular biological methods. Are commonly used fragments of the yellow fluorescent protein (YFP ), 155 amino acids which, or the last (C -terminal ) consist of the first (N -terminal ) 83 amino acids, but also fragments or different fragments of other fluorescent proteins can be used, If to be examined both fragments as a result of the interaction, the fragments coupled to proteins in close proximity to, a collection and storage take place at a complementation Gesamtrohprotein intact, which is capable, after a maturation of fluorescence. The fluorescence can be detected by means of fluorescence microscopy or fluorescence spectroscopy, and quantified.

The degree of complementation is relevant from a physical contact of complementary fragments and thus dependent on an interaction of the coupled proteins to study them. Because once formed BiFC complexes are stable, temporally short interactions of investigated proteins in living cells can be detected. In contrast to the method based on resonance energy transfer FRET and BRET allows the bimolecular fluorescence complementation, due to the slow maturation of fluorescent protein, no time-resolved study of molecular interactions. The position of the study protein fluorescence protein fragment - coupling, which can be crucial for complementation, and the Selbstkomplementationsrate the fluorescent protein fragments that can lead to false positive interpretations shall be considered as limiting for the bimolecular fluorescence complementation of the application.

Variations

Later versions are based on a complementation of fragments of differently colored fluorescent proteins, which at the same time a plurality of protein -protein interactions can be detected ( multi-color fluorescence complementation ). By combining bimolecular fluorescence complementation with FRET or BRET also tri - or oligomeric protein complexes can be detected.