Dear all,
I'm searching for methods to calculate Forster Radii of FRET pairs, ideally for water-soluble and membrane dyes.
Although I am relatively familiar with working with microscopy FRET, I am not familiar with using it to calculate quantitative parameters, the FRET radius being one of them.
I have access to spectrofluorimeters, confocal microscopy and FLIM.
Papers, protocols or anything that might be useful will be highly appreciated.
Kind regards
Dear Rafael Lira ,
Not entirely sure why you want this, but do realize that quite a number of details are already known for a large number of couples, see for a good source:
https://www.thermofisher.com/nl/en/home/references/molecular-probes-the-handbook/technical-notes-and-product-highlights/fluorescence-resonance-energy-transfer-fret.html
See for a good comprehensive overview and/or background theorie behind it:
Article A Guide to Fluorescent Protein FRET Pairs
Article Estimating the distance separating fluorescent protein FRET pairs
and
Müller, S. M., Galliardt, H., Schneider, J., Barisas, B. G., & Seidel, T. (2013). Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells. Frontiers in plant science, 4, 413.
Some good examples in case you want/need to do some work on this yourself:
Padilla-Parra, S., Audugé, N., Lalucque, H., Mevel, J. C., Coppey-Moisan, M., & Tramier, M. (2009). Quantitative comparison of different fluorescent protein couples for fast FRET-FLIM acquisition. Biophysical journal, 97(8), 2368-2376.
or
Article Characterization of the Resonance Energy Transfer Couple Cou...
Hope this helps you a bit further.
Best regards.
Calculating the value of Ro for a FRET pair involves measuring the corrected emission spectrum of the donor, the absorption spectrum of the acceptor, the refractive index of the medium, and the quantum yield of the donor. I agree with Rob Keller that you should not do it if the value can be obtained from the literature, unless you just want to learn how to do it.
Since you mentioned a membrane dye, you have to consider the possibility that the assumption that the orientation factor kappa-squared = 2/3 is false. I was in this situation at one time. Therefore, I refer you to my paper in which I performed the Ro measurement for a FRET pair in which one of the pair was membrane-embedded. The method also involves making fluorescence polarization measurements. This calculation produces a range of values, rather than a single number.
The methods section in this paper goes through the Ro calculation in detail.
Article Extraction of Hoechst 33342 from the Cytoplasmic Leaflet of ...
Please look at the following below links which may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3810607/pdf/fpls-04-00413.pdf
https://www.microscopyu.com/tutorials/f%C3%B6rster-fluorescence-resonance-energy-transfer-with-fluorescent-proteins
Thanks
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes