Bimolecular fluorescence complementation (BiFC) is a method of viewing the association of proteins inside living cells. Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl The cell is the structural and functional unit of all known living Organisms It is the smallest unit of an organism that is classified as living and is often called Hu CD et al. (Mol Cell 2002) solidified this methodology as viable in vivo. In vivo ( Latin: within the living means that which takes place inside an organism.
How it works
The intact Green fluorescent protein (and its variants CFP, YFP, BFP, and RFP) is fluorescent. The green fluorescent protein ( GFP) is composed of 238 Amino acids (26 However, when the fluorescent protein is split into N and C-terminal halves, the molecule does not produce fluorescence. Fusing of each of the two non-fluorescent fragments to two putative interacting partners leads to restoration of fluorescence within a cell by reconstituting the split flurophore. This fluorescence is detected via fluorescence microscopy, which can be recorded by a mounted camera. A fluorescence microscope (colloquially synonymous with epifluorescent microscope) is a light Microscope used to study properties of organic or inorganic substances A camera is a device used to capture images either as still Photographs or as sequences of moving images ( Movies or Videos. The advantage of the BiFC method over other methods of visualizing protein-protein interactions is that it gives an indication of interaction, as well as cellular localization of the complex. Protein-protein interactions refer to the association of Protein molecules and the study of these associations from the perspective of Biochemistry, Signal transduction
BiFC can be used as an alternative to FRET, or it can complement FRET by its possibility of screening protein-protein interactions and their modulators through combination with other techniques like DERB[1]. Förster resonance energy transfer (abbreviated FRET) also known as Fluoresence resonance energy transfer or resonance energy transfer ( RET Dual expression recombinase based ( DERB') single vector system' is a method of efficient cloning and subcloning of Plasmid vectors for high throughput screening (HTS .
References
- Hu, CD, Grinberg, AV and Kerppola, TK. Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC) analysis. Current Protocols in Cell Biology, 21. 3. Wiley Interscience, 2005
- Lu JP, Beatty LK, Pinthus JH. Dual expression recombinase based (DERB) single vector system for high throughput screening and verification of protein interactions in living cells. ". Nature Precedings 2008 <http://hdl.handle.net/10101/npre.2008.1550.2>.
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