Imaging GFP in S. pombe - (Sep/01/2011 )

Hi folks,

So I've been trying to use GFP-tagged proteins to visualize protein localizations in the fission yeast, S. pombe.
We've been trying to image them using a confocal.

The problem I've been having is that my wild-type shows something for the GFP-channel as well!
I've looked at wildtype from obtained from other labs AND commercially bought ones and they all look the same.
I've tried growing them to mid-log phase in YES, EMM, SC, and YNB Complete media. For imaging, I load the cells
onto the slide while they are in the same exact media they were grown in.

At mid-log phase, grown in YNB Complete media, the "GFP" looks like blobs that basically only outline the cell
(probably on the cell membrane) and it just seems to flow in a cycle around the cell.

I've been told this isn't normal for GFP and it's probably aggregates of something that can be seen in the
GFP channel. Currently I'm at a lost and was wondering if anyone else working with yeast has encountered this
problem as well and might have some insight into why this is occurring.


Thanks!

Hey
you said you're loading your cells on a coverslip. Do you coat this and is it possible that this is giving background? We pipet our yeasts on an microscopyglass and put a coverslip on top (not ideal cause our yeasts tend to 'swim). But keep in mind that coating solutions can give background
Good luck

No, I just simply take the tube that I had been incubating O/N and pipet a very small amount of yeast-liquid onto a plain glass slide. Then I place a coverslip on and look under the microscope.

I've been wondering if simply washing the cells and resuspending in fresh media might be a rationale solution-possibility? I'm having a tough time pinpointing possible sources of autofluorescence (ie. ethanol produced from the yeast, strange protein aggregates in solution from lysed cells, strange protein aggregates from secretion, etc.).

UPDATE:

So, I tried again growing the yeast (fission) in YNB Complete media from OD600 ~ 0.2 O/N to mid-log phase (OD600 0.6 - 1.2)
and, even though the nonspecific GFP signal was still there, it seems that the signal from actual GFP was much more intense.

Although I was not able to completely get rid of the background, the real GFP signal was obvious so that'll have to do.
I have NOT repeated this so I do not know if this was the quasi-answer to the problem.