I am trying to visualize under confocal microscopy some GFP tagged proteins after I treated the cells with sodium azide during 30 minutes at 30ºC. I just take the cells at OD 0,4-0,6 in SC media (without Sorensen buffer; I don't know if this could be important) and I add sodium azide to get the culture at 0.5% (v/v). I incubate the culture during 30 minutes. After that, I spin the cells to concentrate and prepare the sample to directly visualize. Do you know if I should add something or I have to prepare these samples in a different way? Is there a special kind of sodium azide to do this experiment? Thank you.
Hi,
Just to make sure, you are trying to see GFP fluorescence in yeast, right?
I have a fair amount of GFP observation in live yeast cells, but never with sodium azide. So I cannot give you a definite answer.
However, I can speculate something based on the known facts:
- SC medium is acidic (If I remember correctly, around 5).
- GFP loose fluorescence under acidic pH
Here's my speculation.
In the presence of sodium azide, cells are depleted for ATP. Therefore, yeast cells cannot maintain the normal H+ potential within the cells using H+-ATP pump. Hence the intracellular pH will be close to that of the medium.
If this speculation is correct, addition of Sorensen buffer may help.
Hope this helps.
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