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.
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-