GFP background reduction

Michael J Conboy conboymj at Stanford.EDU
Sun May 28 16:45:32 EST 2000


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
background).

- Pass cultures while still in log phase growth, and examine cultures
while in early log.  (Reduces vacuolar, and some smaller, yellow-green,
punctate autofluorescence).

- 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,
Mike Conboy
Cyert Lab,
Dept. Biological Sciences

In article <8grkmg$9pn$1 at mercury.hgmp.mrc.ac.uk>,
Liam Good  <good at biobase.dk> wrote:
>
>Hi,
>
>Any advice on how to reduce background autofluorescence when using GFP as
>a reporter in yeast?
>
>Liam
>
>
>---
>
>




______________O_________oO_____________oO______o_______oO___________________
YEAST bionet newsgroup see: http://www.bio.net/hypermail/YEAST/
YEAST e-mail: messages sent to yeast at net.bio.net
subscribe: e-mail biosci-server at net.bio.net with: subscribe yeast
unsubscribe: e-mail biosci-server at net.bio.net with: unsubscribe yeast
YEAST on the WWW: http://genome-www.stanford.edu/Saccharomyces/VL-yeast.html
problems with the YEAST newsgroup? E-mail the moderator: francis at cmmt.ubc.ca







More information about the Yeast mailing list