Labeling of my protein with donor is 1 while labeling of my protein with acceptor is 0.8. Donor and acceptor are present on different set of proteins and I'm trying to look at the interaction between these two proteins. I'm working with A488 and A647.
I'm getting higher intensity of donor (in D+A mixture) as compared to donor alone. This gives me a negative FRET efficiency. Kindly let me know where the problem lies.
Look at the stoichiometry and analyze only the signals where you have both donor and acceptor present. Stoichiometry vs. FRET plots allow you to distinguish between donor-only, donor-acceptor, and acceptor-only species. You might have a look at the attached publications. They deal not only with stoichiometry, but they use it. Good luck!:)
Article Single-pair FRET characterization of DNA tweezers
Article Pulsed Interleaved Excitation
I would suggest two modifications to the protocol: First Alexa 488 and 647 have a very low spectral overlap. This will make FRET efficiency quite low. I would change from Alexa 647 to Alexa 568. This will give you much higher FRET efficiency. Secondly I would use the sensitized emission approach (acceptor bleaching). This method gives more reliable results for low efficiency.
You can easily design a positive control experiment: label the same primary antibody with two secondaries at varying ratios (e.g. 1:2 1:1 2:1). Because both secondaries attach to the same primary you will get optimum FRET efficiency for your positive control. The control will also let you optimize you imaging approach without wasting precious experimental samples.
I hope this helps. Have fun and best of luck!
If you are seeing higher fluorescence intensity of donor in the presence of acceptor than in its absence, you are probably getting some acceptor fluorescence at the donor emission wavelength. Measure the fluorescence of the acceptor in the absence of the donor at the donor emission wavelength and subtract it from the fluorescence of the mixture.
Forgot to mention: if your acceptor labeling stoichiometry is only 0.8 instead of 1, and the binding stoichiometry is 1:1, and assuming there is no difference in affinity of the donor protein for labeled and unlabeled acceptor protein, the energy transfer efficiency will be only 80% of what it otherwise would be if the acceptor labeling stoichiometry was 1.
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