Does anyone have experience trying to work around background fluorescence in cyanobacteria?
I'm currently trying to image a GFP fusion protein, but I didn't realize there was significant background fluorescence in the green spectrum (phycoerythrin I think).
I'm looking for a way to decrease expression or fluorescence of phycobiliproteins while maintaining normal expression/function of other things? I'm going to try keeping my bacteria in the dark for a couple of days before imaging (hopefully lowering phycobiliprotein expression) but I'm concerned this might have a more widespread effect on expression or overall physiology than desired.
You could get a broad band filter to cut off your background fluorescence from a commercial source.
Hello!
Although both compounds are excited at similar wavelength, they do have different emission maximum. A bandpass filter within 490-500 to 540 nm should solve your problem.
Phycoerythrin: emission ~ 540 to 600 nm, em maximum at 573 nm;
GFP: emission ~ 480 - 570 nm, em maximum at 510 nm
You can try and do an extraction to remove the protein and quantify that way. You will get background flouresence as well as absorption of light from other pigments in the organism. That being said, If you are comparing relative fluorescence it shouldnt be a problem if you compare it with a non-expressing culture.
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