I need to perform ChIP on isolated nuclei, but i don't know if I should use 1% formaldehyde or if I should decrease the crosslinker concentration?
We use the same way to perform DNase seq but without crosslinking.
Thanks a lot for answer i will try different conditions.
Dear Jean-Clément,
I was wondering about doing the same, isolating nuclei and then doing Chip. The reason is that my protein is also abundant in cytoplasm and I would prefer enriching the nuclei and then cross linking. I am curious to know what your findings were and whether you would recommend crosslinking just the nuclei.
Dear Linda,
After trying different condition i conclude than fixation don't change after nuclei isolation. I just compare condition between isolated nuclei and entire cells after sonication and DNA looked similar.
But in your case why don't you purify your nuclei after fixation? It's will remove most of the cytoplasm contaminant?
Thank you Jean-Clément,
The problem is that my protein is very very abundant in the cytoplasm and very lowly expressed in the nuclei. I tested cells that I did not crosslink and cells that I crosslinked (by western blot- blotting for my protein and other cytoplasmic/nuclear markers) and it seems like I get a lot of cytoplasmic proteins when I crosslink (ex my protein and tubulin), [ the fractination of crosslinked cells is not complete, things like cytoskeleton, and proteins bound to it still remain stuck even after fractination]
I want to do Chip on this protein so I want to maximize the amount of nuclear protein that I get. Otherwise my antibodies will mainly bind to cytoplasmic protein. Therefore isolating nuclei and crosslinking after seems like a solution to this problem.
Did you still use 1% formaldehyde [RT or cold?] for 10min? in PBS? and the amount of sonication needed was not affected that much? [compared to fixing whole cell]
I will need to test it as well but just wondering about the exact protocol you ended up using.
thanks
Linda
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