Hi, everybody. I'm preparing FRET AB experiment and at the moment my only available pair is YFP-RFP. I'm fully aware it is not common donor-acceptor pair as EGFP-YFP, CFP-YFP or CFP-RFP, however, according some of the literature out there it should be possible. Nevertheless, I would appreciate if you could give me advice how to approach the experiment (troubleshooting) in order to separate donor-acceptor fluorescence since the fluorescence emission spectrum of YFP is red‐shifted when compared with EGFP and its spectral overlap with the RFP is greater. I'm planning to use Leica SP8. Moreover, I would appriciate further feedback about EGFP-mKATE as potential FRET AB pair.
As a rule of thumb, larger spectral overlap will yield a longer R0, therefore your FRET is going to be sensitive to longer distances. What is the expected distance to measure (including the distances to the FP chromophore centers)? What is the R0 value. Compare using the Forster Relation and see whether you stand a chance of observing the required result. Take into account that the spectral crosstalks are also higher therefore you need to carefully select the wavelength range at which you collect fluorescence.
Are you performing inter-molecular FRET whereby you have an FP per each macromolecule and see FRET upon binding of two macromolecules or intramolecular FRET where you have both FPs on one macromolecule and observe a change in FRET efficiency?
If you want to follow the dynamics of the process, indeed you need to separate well the signals of YFP and RFP, as you will need to follow the signal over time. But if you want just 'a picture', on fixed samples, to see if there is FRET or not, I would just try acceptor photobleaching. Just acquire an image of YFP with a low power (or a few images to get an average value), then bleach the RFP (strong power with 532 or 561), then take again an image (or a few) of the YFP. If there is FRET you should see an increase in the intensity of the donor.
Controls:
- check that the bleaching of RFP is complete
- using the conditions necessary to bleach RFP, check if the intensity of the YFP remains the same before and after (on a sample with only YFP)
- ideally you should have a positive control (that you know there is FRET) and a negative control (that you are sure there is no FRET), but which control should that be depends on your experiment.
mKate is not very bright, I would advice to stick to some red FP, mCherry, RFP, mRuby...
Thank you, Eitan and Susana.
Eitan, I'm preforming inter-molecular FRET. When it comes to the molecular distance - for a start - I'm dealing with two STIM1 proteins that are known to form oligomers due extension during conformational change. Hopefully this also answers to you, Susana; I'm using two proteins of known interaction (positive control). My RFP and YFP tags are on the same side of the membrane. For a negative control, I was planning to use STIM1 proteins with tags on different sides of the membrane
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