Hello everybody,
I would need some tips regarding the selection of an optimal FRET donor/acceptor (D/A) pair for labeling proteins site-specifically and detect their potential interaction via FRET-based assay on a solid support (e.g., microplate, chip). As I mentioned, I would use a fluorophore-maleimide reagent since my recombinant proteins are engineered to contain C-terminal free cysteines but I have problems with picking the most optimal pair of fluorophores.
1. I am designing an assay on a solid support (on the surface of a microtiter plate at the beginning) where each interacting protein (one immobilized, other free) would be labeled with 1 (or less) fluorophor (donor or acceptor) per protein. Which of the characteristics of individual dye pair (e.g., Förster radius (Ro), quantum yield, ext. coeff., spectral overlap (Stokes shifts)) are the most important to take into account in my case?
2. In one of my assay setting the predicted distance between D and A is ~50 Å, so I am looking for a pair with relatively large Ro to achieve FRET efficiency greater than 50%. Am I right to pay so much attention to Ro? For example, for reported Cy5/Cy5.5 pair Ro is ~80-85 Å but I am worried with excitation and emission spectral overlaps (http://www.olympusconfocal.com/theory/images/fluorophoresintrofigure6.jpg) and consequent "bleed-througs".
3. In another setting the distance between fluorophores is predicted to fall into 20-80 Å range (depending on complex conformation), so I am looking for a well-functioning FRET dye pair with Ro~40-50 to cover the dynamic range. Any suggestions?
4. In literature (http://www.ncbi.nlm.nih.gov/pubmed/20103093) I also found DY-481XL/DYQ661 pair as interesting because of the large Stokes shift of donor (~125 nm) but have not been able to find a Ro value for this pair - does anyone have such information?
I would be very grateful for every helpful advise and answer to any of my questions.
Thank you,
Peter Molek
It might be helpful to refer to the literature on single-molecule FRET studies of biomolecules, since these use surface-immobilized systems.
Cy5 and Cy3 are very insoluble and difficult to work with. 5FAM the stable version of fluoresce in, and Dabcyl are very cheap and quite useful. But I believe fluoresce in dyes are pH dependent so this may be an issue for you if you use multiple pH conditions. The Alexa dyed are quite expensive and are not off patent.
Hi does viscosity or addition of polyethylene glycol (at same pH) also affect FAM fluorescence?
Fluorescence polarization is affected by viscosity, increasing with increasing viscosity of the medium. I don't think fluorescence intensity is affected by PEG itself, unless the concentration is so high that it significantly changes the concentration of water. In that case, there might be an increase in fluorescence intensity. Contaminants in impure PEG might cause quenching of fluorescence if the concentration is high enough.
Thank you Mr. Shapiro. I have used a maximum of 40% w/w of PEG 200/2000/6000 in phosphate buffer. I have seen a very small increase with decreasing molecular weight but I guess that can be neglected.
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