I am interested on obtaining native chromatin from Hela cells. The ultimate goal is to do mass spectrometry in the purified fragments in order to check the proteome found there. Yet I am wondering on how to get chromatin DNA as native as possible without losing too many proteins in the process. I have thought so far on maybe lyse the plasma membrane and nuclear membranes (how?) and then from those extracts do column purification using maybe histone antibodies. would that be a good way, is there any better possibility?
Inigo,
The methods in the publication below should be a good starting point.
All the best,
Article Argonaute proteins couple chromatin silencing to alternative splicing
There are a couple of published protocols that are helpful - i have attached a few. Also refer to the 2013 paper (Voigt et al) by Garcia and Weinberg laboratory where they map combinatorial modifications on histone peptides, because they isolate chromatin in its native state examine it through tandem mass pec. analysis of different sorts to answer a similar question that you would like to. hence I think this paper would be most relevant.
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