If there is intramolecular FRET occurring, the intensity and lifetime of the fluorophore will be lower than those of the free fluorophore. This may not be conclusive, however, because the fluorophore may have lower quantum yield and lifetime just because it is attached to the protein and interacts with it.

You could look for concentration-dependent oligomerization of the protein by measuring the effect of its concentration on its fluorescence. As long as the inner filter effect is insignificant or corrected for, and the fluorescence intensity remains in the linear range of the detector, oligomerization resulting in intramolecular FRET will result in a sublinear increase in fluorescence with concentration. You can use sub-optimal excitation and emission wavelengths to increase the range of testable protein concentrations.

Two fluorescence-independent ways to determine if your protein is self-associating or aggregated, as opposed to monomeric, are (1) dynamic light scattering and (2) size-exclusion chromatography-multiangle light scattering (SEC-MALS). Both methods require special equipment.

If concentration dependent, should it be intermolecular FRET?

Intramolecular FRET occur when the protein labelled with too many fluorophores/fluorophores labbled too close, I think!

Can you provide more details about your system?

Intra-molecular FRET will occur independent of concentration.

Inter-molecular FRET depends on Förster radius and and concentration.

Assuming point like molecules, you can get a distance distribution from the concentration, see e.g.:

https://en.wikipedia.org/wiki/Mean_inter-particle_distance

If you have your Förster radius, you can calculate a theoretical Förster efficience vs. Distance curve.

Now multiplying the 2 curves gives you the actual FRET in solution distribution,

Summing this up /integrating gives you the amount of background FRET.

e.g. If you have R0=6 nm and 100µM sample concentration you will up with

~ 7.6 % FRET as background. ( see png's, I can share an xls if needed).

However avoid doing FRET at such high concentration: at concentration above a few µM. You will have 2 other effects to deal with:

1. internal Filter. Concentrated Fluorophore solutions will absorb ( by Radiation, not by FRET) you Fluorescence light. This depends on layer thickness ,absorption and concentration (Lambert-Beer).

2. Cross-talk and direct excitation. Usually you have than one Partner at high and one at very low concentration. In this case only a tiny Fraction of the signal will be actually FRET and not X-talk or direct-ex.

cheers

Ralf

PS.: This is of cause all over simplified. as HOMO-FRET, kappa-squared etc is ignored.

I agree with David, more info would be nice. If you’re getting unwanted intramolecular fret then you need to redesign the fret sensor or reformulate the solvent or both.

Dear Xupiter Monk ,

I am afraid you provided too little information. Be fully clear about the use of the term intra-molecular as indicated by Guobing Xiang and Ralf Schmauder . See also figure (from: https://slideplayer.com/slide/4627800/ ).

Questions are:

-Are you really talking about intramolecular FRET?

-Are there two different labels inside one molecule involved or one label at multiple sites?

-What type of protein are we talking about, is it large and can circumstances be created were it is highly folded or unfolded?

-Etc. etc.