Hello,
Have you ever extracted nuclei from fixed samples ?
When I fix my HepG2 cells with 1% formaldehyde, I noticed that the fixation prevents cell lysis with regular nuclei isolation buffer, syringe (26G) or dounce.
Thank you in advance for your help!
Hi, I find it impossible to get pure nuclear isolates from fixed cells (I use 2% formaldehyde). Most proteins become crosslinked to, I assume, the cytoskeleton and nuclear envelope and are centrifuged out with nuclei when performing nuclear isolation. Can you perform the isolation and then fix the isolated nuclei instead? What are you going to do with obtained fixed nuclei?
Hello,
Thank you for your answer!
No unfortunately I cannot do that as it can lead to changes in protein-DNA interactions (I want to perform ChIP experiments). My boss and I think it is crucial for what we want to do (which I cannot discuss in details) to stay as close as possible to physiological conditions while doing cross-link :/
Did you try the standard ChIP protocol? Why would you like to isolate nuclei for the purpose of ChIP?
Because we work on virus infected cells and want to ChIP the virus form that is in the nucleus and not the intermediates that are in the cytoplasm (they are almost identical and cannot be discriminated by PCR or sequencing), which is very complex.. We used the standard ChIP protocol before but it doesn't allow us to determine which form of the viral DNA is bound by the proteins.
That is a very specific operation. I do not think that ChIP will help you with that.
Improvement that could potentially work is a reduction in formaldehyde concentration used for crosslinking - I would try 0.01, 0.001, and 0.0001% and no longer than 5 min.
I've attached an image of how formaldehyde crosslinks proteins to DNA in 15 min of treatment. As can be seen for the 0.1% treatment almost all proteins were isolated with the DNA.
I would also try isolating nuclei first and then fixing them (I know you said that this would not be preferred) but it will give you a clear cut-off: cytoplasm/nucleus.
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