It would help if you would describe the specific background you are
experiencing. Pre-empting that, here are some things that worked for me:
- Supplement ade2 stains with twice the adenine used in synthetic medium.
Add adenine to rich medium also. (Reduces vacuolar broad-spectrum
- Pass cultures while still in log phase growth, and examine cultures
while in early log. (Reduces vacuolar, and some smaller, yellow-green,
- Reduce both the field diameter and intensity of your excitation
illumination, if practical.
- Reduce expression of your GFP construct, if practical. (Reduces
non-specific localization and potential fluorescence from
partially-degraded or cleaved GFP fusion protein, or so I suppose).
- Use a different strain background. I found the YPH and SEY6211 strains
to have a lot of vacuolar autofluorescence, which was fortunately quite
manageable by adenine supplementation, and little other autofluorescence.
An SF838-xxx strain gave a punctate, peripheral, dim yellow-green
fluorescence, which was reduced in early-log cells. A DBY4974 strain
(ADE2) appered to have very low background across the spectrum.
- Use a filter set with a narrow-band excitation filter at the higher
excitation frequencies, and a narrow band-pass emitter.
Hope this helps,
Dept. Biological Sciences
In article <8grkmg$9pn$1 at mercury.hgmp.mrc.ac.uk>,
Liam Good <good at biobase.dk> wrote:
>>Any advice on how to reduce background autofluorescence when using GFP as
>a reporter in yeast?
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