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Imaging GFP in S. pombe


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#1 TVo

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Posted 01 September 2011 - 07:45 AM

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!

#2 fysio lab

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Posted 02 September 2011 - 12:03 AM

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

#3 TVo

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Posted 02 September 2011 - 11:53 PM

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.).

#4 TVo

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Posted 12 September 2011 - 08:11 AM

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.




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