Nuclei isolation for Chromatin IP - (Feb/14/2008 )

Hi everybody,

I am looking for a protocol dealing with nuclei isolation for Chromatin Immunoprecipitation. I am using transfected 293 cells and want to get rid of all the "cytoplasmic waste".
There are some protocols where people do not x-link their cells but the isolated nuclei...?
Which detergent should one use? Triton-X 100 or NP-40? and which concentration?

Thanks

---

I lyse my cells in 10mM Tris pH 8, 10mM NaCl, 0.2% NP-40 (added fresh on the day) + protease inhibitors. After spinning down, removing the supernatant will get rid of the ~cytoplasmic waste~. Then I lyse the nuclei in 50mM Tris pH8.1, 10mM EDTA pH8.0, 1% SDS + protease inhibitors and proceeed to sonicate.
Hope this helps

---

Thanks Clare for your help,

what do you or anyone else here think about crosslinking the nuclei after having prepared the nuclei ?

---

I personally wouldn't, as lysis steps etc are likely to dissociate proteins from DNA

---

When lysing the cells after crosslinking, you get rid of most of the membranes and soluble proteins. What remains is the nuclear lamina, chromatin, associated cytoskeleton, and remnants of the golgi and ER which are associated with the cytoskeleton and nucleus. Are these things a problem for what you're looking at? If the things you are looking at are likely to be soluble in the cytoplasm then you don't need to worry about isolating the nuclei before crosslinking.

Also, I agree with Clare that isolating nuclei first may create some problems. Part of the point of crosslinking is that you lock everything in place before isolating the nuclei so you don't get any redistribution of factors during this process. If you crosslink after isolating your nuclei you may lose some interactions between protein and DNA and gain others that aren't normally there in vivo.

---

Thank you, very much.

The problem i have is that i transfect a fusion-protein with a tag into my 293 cell line. Untill now i have no cell line stably expressing my fusion protein. So at the moment I'm fighting with high background problems due to the transfection...

---