Hey!
I have a FRET system between the inner and outer nuclear membrane, and I was wondering if I stained with DAPI or any other fluorescent antibody if that would mess up my FRET readout? As of now I am using acceptor photo bleach and I am working with neuroblastoma cells (Be2-c). I can do either fixed or live cell imaging, depending on which would work best if this even possible.
Thank you!
I would expect the use of a fluorescent stain such as DAPI or a fluorescent antibody to interfere with your FRET measurements. Best avoided.
It will depends of your FRET design. If a fluorochrome such as DAPI is not emitting or absorbing the light wavelength needed in your FRET system you should not have any problem. However, DAPI is a fluorochrome with a very broad spectrum of absorption and emission so it is maybe not be recommended. You could use phalloidin coated with an Alexa fluorochrome that is not having interference with your FRET system.
You can check the fluorochrome spectra of individual dyes and their overlap with other (if any) in:
https://www.thermofisher.com/order/fluorescence-spectraviewer#!/
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