Question

How do transfections work? And how does FRET work? Like if I had a protein called X lets say that I wanted to analyze the function of, how would I do that using FRET? I dont understand what FRET is or how it works...doesn't it just tell you the location of a protein?

And how does inserting this protein into a plasmid and then inserting that plasmid into a cell help you study this? I am so lost.

Answer 1

Transfection is a process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.Intaking of the nucleic acid by cell is done by endocytosis.This nucleic acids are transferred by means of plasmids or siRNA or short antisense oligonucleotides.That is a desired foreign designed DNA insertion in a cell.

FRET is Forster Resonance Energy Transfer. This is a mechanism of energy transfer between two light sensitive molecules ( chromophores). A donor chromophore , initially in its electronic excited state may transfer energy to an acceptor chromophore through nonradiative dipole dipole coupling.The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor ,making FRET extremely sensitive to small changes in distance.Measurement of FRET efficiency acn be used to determine if two flurophores are within a certain distance of each other. This can be done by fluoroscence as well as phosphoroscence. To analyze the function one need to label an experimented molecule as donor and another as acceptor. Then by monitoring FRET efficiency of the complex formation , the interaction or conformational change can be estiamted.For example - protein protein interaction , protein - DNA interaction , protein confomational change etc.

It does not only locate a protein but by this technique one can observe the FRET change when there is a change in distance and relative orientation of donor and acceptor due to the twist or bend occuring. This technique als helps to find out ligation binding of protein.One can get information about an unknown protein's conformation by FRET.