I would like to use FRET effect to probe the distance between two functional moieties (I am sure the distance is smaller than 10 nm). I have tried several times, but I found out that the FRET efficacy of some groups were larger than 95%. I think this was not resonable enough. Could someone tell me the proper range of FRET efficacy?
There is an equation that relates the FRET efficiency E to the distance between a single donor and a single acceptor (r) and the parameter Ro, which is a characteristic of the donor-acceptor pair and a few other aspects of the system.
E = 1/(1 + (r/Ro)6)
Since values of Ro of organic donor-acceptor pairs are not usually more than about 6 nm at best, the FRET efficiency for a pair separated by 10 nm is going to be very small, only a few percent.
If r=Ro, then E= 0.5, i.e. 50% efficiency.
The calculated value of Ro includes an assumption of a random orientation between donor and acceptor dipoles. If this assumption is not true for a particular system, the value of Ro may be incorrect. This could happen, for instance, if one of the pair is immobilized, such as being intercalated into DNA or a lipid bilayer, or bound in a protein binding site.
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