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Formaldehyde cross link after washing cells - (Aug/19/2007 )

Hi,
I am working with yeast and I have been advised that before cross linking, I should pellet cells, wash them with water and then crosslink. By this means, I can get rid of titration of formaldehyde by the enormous amount of amine groups in the media. I think it sounds logical but i could not be sure.
could you think of any possible drawbacks of this method? Also I am doing 45 minutes at RT fixation but to my reading in this forum, it looks a little longer.
Does anyone have any suggestions?
thanks.

-ehallacli-

QUOTE (ehallacli @ Aug 19 2007, 02:40 PM)
Hi,
I am working with yeast and I have been advised that before cross linking, I should pellet cells, wash them with water and then crosslink. By this means, I can get rid of titration of formaldehyde by the enormous amount of amine groups in the media. I think it sounds logical but i could not be sure.
could you think of any possible drawbacks of this method? Also I am doing 45 minutes at RT fixation but to my reading in this forum, it looks a little longer.
Does anyone have any suggestions?
thanks.


In our lab we fix in the media for 20min at RT. 45min sounds a bit long.

-KPDE-

we fix cells for 10 min RT with 4% PFA but after washing the cells in PBS.

-scolix-