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FRET only works for interactions between 1nm to 20nm.

How can you be sure that the interaction that you want to study isn't less than 1 nm/greater than 20nm apart?

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To get an elaborate answer on this one, I guess you should ask at physics. Very basically, FRET works by that an excited chromophore transfers energy to a nearby chromophore, through dipole-dipole coupling, which in turn emits energy (light) which can be detected. This is a quantum mechanic effect, i.e. these are very small energies. The emission energy between from the donor to the acceptor chromophore is inversely proportional to the distance between to the power of six according to the equation:


Where r is the distance and R0 the Förster distance between the donor/acceptor-pair (distance which the energy transfer efficiency is 50 %).This means that the energy to be detected very rapidly decreases. The Förster distance is in the range 2-10 nm for different chromophore pairs, which leads to the upper limit of FRET use.

The lower limit I don't know if it's due to steric effects or some quantum mechanical effect I do not know about. But since proteins are in the size range of the lower limit they must be in really close vicinity in order for their interaction to be detected using FRET.

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The answer is to perform a FRET experiment.

FRET is most commonly used to find out whether something is nearby something else in this range. Its chosen as a technique when two points of attachment for FRET probes might be within 20 nm, but are not known to be, or might be only in certain conditions.

There are some rules of thumb though. 1 nm, or 10 Angstroms, is about the length of 6 chemical bonds. To be that close the two FRET probes (which are fluorescent molecules) would typically be attached to the same molecule. 3-4 amino acids apart in a peptide, at the opposite ends of a steroid. These are schemes for a FRET experiment you would typically avoid out of hand.

FRET is often used for proteins, which are often wider than 20 nm. For instance FRET probes might be attached to specific positions in two proteins which are known to bind to one another. A FRET signal would indicate whether the two attachment points of the FRET probes are physically in proximity, allowing one to narrow down the orientation of the two proteins which are binding.

So its more or less an educated guess as to whether to do a FRET experiment, but using simple spatial reasoning you make a safe bet.

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While this is true, FRET is not a fool-proof experimental approach, and can give either false positives or false negatives. I think Bioguy wants a way to confirm FRET results. – user560 Jul 3 '14 at 19:33
good point. FRET is pretty hard to do.. its not my favorite sort of evidence. its used in so many contexts - we're not necessarily talking about proteins here for instance - that its hard to go too deep... – shigeta Jul 3 '14 at 21:51
That's also true, I assumed the discussion of proteins. – user560 Jul 4 '14 at 0:52

I'll add on to shigeta's and RickardSjogren's answer. Firstly, I agree that the upper limit for Forster interaction distance is ~ 10-11 nm due to the rapid fall off of the effect. If I infer from your question correctly, you seem to be asking how to confirm a lack of FRET interaction is a result of no interction, or steric hindrance.

Classical methods of confirmation can be learned from biochemsitry. For instance, co-immunoprecipitation and intermolecular linkage with pull-down.

If the FRET interaction doesn't show a positive result, perhaps it's because the interaction is so tight and the probes are so close that they are prohibited from the FRET effect. That would suggest a strong interaction, and a co-immunoprecipitation should readily show you that you can pull down one protein, run the results on a western blot, and probe for the other protein. The second permutation is to pull down protein B, and probe for protein A. This is a confirmation that both proteins exist in complex. Failure to co-immunoprecipitate both proteins (especially when trying both permutations) would likely indicate that proteins A and B do not interact, or that their interaction is highly transient or very weak. Co-IP can be demonstrated in vitro using purified recombinant proteins, or in vivo, in their native state from cell lysates.

Enter the second technique. Various small molecule compounds of different molecular length can be used to make inter-molecular, amine-to-amine linkages. See BS3 for example. This works best in vivo, where incubation with a compound such as this would create cross-links between neighbouring proteins only with the reach of the englth of the compounds. If two proteins interact, then by definition they must be spatially close to each other, and therefore some percentage of your proteins should be in complex together. From there, you can run a western blot and show that you can probe for both proteins, that run at the same apparent molecular weight defined by the sum of both proteins molecular weights (and therefore both proteins will run higher than expected because their mass is modified by the addition of the interacting protein + linker). Modern versions of this approach involve using mass spec identification of cross-linked fragments, which is much more sensitive and quantitative that western blotting.

A third method could be quantitative colocalization by immunofluorescence microscopy. Prepare cells for immunofluorescence, probing for both proteins of interest, and quantitatively assess whether the proteins co-localize with each other. By eye (and not so rigorously), you would expect that both proteins should exhibit very similar spatial distributions within the cell if they are in fact interacting.

Failure of the above two methods strongly supports the idea that the proteins do not interact, or that they do interact in very weak or very transient conditions. The only way to truly exclude the idea that they do interact in a very weak/transient manner is if you know that the protein complex does something in a cell. If they do something while in complex, then you can knock one protein out, or express a recombinant mutated form of the protein, and assay for the lack of the normal action.

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