I noticed people use different lysis buffer to extract chromatin in ChIP assay(cross-linking). Do salt and detergent help to dissolve chromatin?
Some paper pointed out mononucleosome still show low solubility in 150mM salt.
Any ideas to improve the solubility of chromatin after sonication?
Thanks,
Weiyan
hello Weiyan,
what do you mean with "to extract"?
If you mean how much chromatin is in solution after sonication: in my personal experience, loads of nuclei show very little insoluble pellet if the sonication is well performed (even after heavy crosslinking, i.e. for CTNNb1 ChIP).
If, instead, you want to know how much is the chromatin after the IP, normally high SDS (1%) and low pH (NaHO3, 50mM) release efficiently all the chromatin fragments from the beads and maintain them in solution, ready to be precipitated/purified.
What is the target of your ChIP? According to different protein you want to precipitate (histon marks rather than transcription factors), you might have to modify the standard protocols to obtain the best performance for your specific IP.
best
Fil
Hi Fil,
Thank you very much for your answer!
My question is If the solubility of nucleosome (chromatin-protein complex) is very low after sonication in low salt buffer, and most of the part will go to pellet after high-speed centrifugation, then how can you expect to get a good IP results.
Actually, I checked the total DNA and protein (my target protein, by WB) in the supernatant and pellet after sonication and found that there are still tons of DNA and proteins in the pellet.
Best,
Weiyan
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