My system is made of a protein that binds to the membrane, which carries the epitope tag, and a nanobody, binding this tag. Membrane binding protein is a fusion protein consisting of a protein fluorophore mNeonGreen and a membrane-binding domain. The nanobody is labeled with Cy5 maleimide. Separately, both protein and nanobody work well in other systems. However, the addition of nanobody to the wells with protein bound on cell membrane leads to a drop in fluorescence in the FITC channel by order of magnitude. I suggest that this phenomenon may be either due to the unbinding of the whole protein complex from the membrane following nanobody binding to epitope tag or due to the mNeonGreen fluorescence quenching (however, as far as I know Cy5 does not quench mNeonGreen). I feel stuck as I don't know how to design a workflow to distinguish between the two.
If anyone has some experience in the field and can share an idea or outline the direction, any advice would be deeply appreciated.
I don't think dynamic or static quenching is likely in this case because the fluorophore of GFP-related fluorescent proteins is buried within the structure of the protein and therefore inaccessible to external collisional quenchers.
Fluorescence resonance energy transfer (FRET) seems unlikely to cause a large decrease in mNeonGreen fluorescence because of the poor spectral overlap between the emission spectrum of mNeonGreen and the absorption spectrum of Cy5. However, if the distance between the mNeonGreen fluorophore and the Cy5 on the nanobody is short, and if more than one nanobody binds to each mNeonGreen, FRET might be a significant contribution.
If you think the nanobody is causing the mNeonGreen-fusion protein to dissociate, see if the loss of fluorescence occurs when you use an unlabeled nanobody. If not, then I'd suspect FRET is occurring.
Adam B Shapiro thank you very much for your answer! Adding unlabeled nanobody to the system was my plan (I just don't have it right now but I can produce it in rather quickly). This experiment eliminates resonance quenching theory. I had some doubts about whether I need to prove the abscence of static or dynamic quenching. However, I could find no data on protein fluorophore quenching except for "classical" data regarding pH, amines, bromide, iodide, oxygen, halogens and some metals so it seems that maybe I don't need to prove it separately.
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