I am trying to set up a direct FRET experiment in Beas2B cells to investigate the interaction between two proteins A and B. I set up three dishes, namely CFP-A, YFP-B (controls) and CFP-A + YFP-B.
However when I excited and collected CFP-A dish with YFPex and YFPem, I was able to see very strong signals (as attached). These signals do not seem to be autofluorescence as they are particularly strong in certain cells. Indeed, the fluorescence signal coincide exactly with the image I saw with CFPex and CFPem.
Vice versa, when I excite and collect YFP-B dish with CFPex and CFPem, there was also weak crosstalks.
Could anyone suggest if these strong signals are due to autofluorescence or perhaps mis-setting of the confocal microscope? Is it possible that a CFP tag would give YFP signal?
Thanks a lot in advance!
Hi Sam,
It doesn't look like autofluorescence it looks like there is something wrong in the settings, you can check your settings using the spectraviewer tool from Life Tech
https://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer
Try an untransfected dish and if you get the same it is autofluorescence.
CFP tag should give some emission in the yfp channel when exciting for cfp.
You would not necessarily expect so much light from YFP cells when exciting for cfp but it is possible. The use of the nF-FRET calculation method takes these cross-excitation and emission bleedthrough constants in to account.
This paper is good to browse for an idea how to utilise these calculations : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491707/
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