Is it possible to fix FRET biosensors such that after fixation the readout is still true, at least relatively? I understand the efficiency of the FRET may be weakened, but is the readout still accurate?
Jordan, we usually fix our samples prior to looking for FRET readout (it makes it easier so we don't have to account for moving parts). We looked for FRET first with a spectrofluorophotometer and saw the same effect with our fixed samples. We designed a negative and positive control as well to make sure this wasn't something random.
Article Fluorescence Resonance Energy Transfer (FRET)-based Detectio...
Thank you for your response!
You observed a FRET inter-molecular interaction at the ruffling edge of membranes in MDA cells, which, with sufficiently expressed protein, should be apparent in live or fixed samples. Did you notice or measure if there was a decrease in response or efficiency? I am using a FRET biosensor (two FRET members linked by a bio-responsive peptide) and am concerned whether or not the fixed product will be representative of the the true concentration of measured analyte (to which the bio-responsive peptide responds).
We did look at this with live samples as well. We used the fixed approach because it became difficult with changing plane due to cell movement, however, we were able to readout some response for FRET in live cell. With our system, we detected a pretty robust efficacy signal (~20%) which was actually higher than in the live cell system we tried to use.
How would you explain the change in efficiency between the live and fixed samples? My concern is that I will get an artificial signal change upon fixation which I will read as a real response to my intracellular analyte concentration.
Hi Jordan, Did you solve this yet?
I'm in a similar boat; but was thinking if you have an experiment where you have positive controls that elicit say 10%-100% FRET signal as a dose response, in live cell and apply these same conditions to the fixed you might be able to get a handle on how much fixation changes the output. I'd imagine this will depend on what you are trying to detect and what your sensor is based on though.
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