Hi all,
I want to perform sequential ChIP. However I have a setting where I can't end up with a big volume of diluted eluate after the first round IP. Also the big volume reduces the efficiency of the second IP. So my Idea is to use Ampure beads to get out the chromatin from the eluate such that I don't end up with such a huge volume. Does anybody know if Ampure beads can be used for Chromatin, so not pure DNA itself. Thanks.
Hi Rutger,
Rather than having an answer to your question, I would suggest an alternative: use Amicon Ultra filters. These columns are specifically made to concentrate (and desalt) small samples without any loss of material and can be used in a standard mini centrifuge. The Amicon Ultra 0.5 mL Ultracel 30k (Millipore - UFC5030) can be used for DNA fragments from 100 - 500 bp (see e.g. supplemental protocol in http://www.ncbi.nlm.nih.gov/pubmed/22652625).
Like it, I would say not. 1st I'm not sure that chromatine will be able to bind. 2nd, the nature of TF bound on the chromatin could impact the binding to beads (if there is binding). 3rd; you need to wash with ETOH that may kill epitopes...
So, a lot of variables... If you want to do, you'll need to perform a lot of control to be sure that it is working fine. But like it I wouild say, no...
Good luck
@Daan, Thanks for the tip. looks pretty solid. however one more thing. Since I want to end up with enriched DNA bound histones would'nt it be better to use 10K collumns instead of 30K?
thanks
Hi Rutger,
For your application, I think there's little difference (see link below).
You should use a pore size that doesn't let your crosslinked DNA-histone complexes pass. Considering that DNA > 100 bp doesn't pass the 30K filter, you should be fine for your complexes as well. But, 10K filters should work equally well. The difference would be made if you want to get rid of certain small peptides and oligonucleotides, with 30K filters letting pass somewhat larger fragments than 10K filters.
https://www.merckmillipore.com/GB/en/product/Filtres-%C3%A0-centrifuger-Amicon-Ultra-0,5-ml-pour-la-purification-et-la-concentration-de-prot%C3%A9ines,MM_NF-C82301?bd=1
In the following paper, the authors use Ampure beads for chromatin 'purification/enrichment'. Nature Methods 12, 71–78 (2015) doi:10.1038/nmeth.3205
However, in my hands chromatin/nucleosomes (as well as some cellular proteins) got 'irreversibly' bound to the beads and it was impossible to get them back into solution when TE, ddH2O or buffer containing moderate conc. of salt & non-ionic detergents were used for elution. The eluate from the beads contained only DNA with no/very low protein content. My chromatin fragments were bigger than what described in the paper and prepared from a different cell line, though.
So, as others suggested, it may be better to go for Amicon or Micron device. Or using input/bulk chromatin, I recommend to test if your chromatin can be released from Ampure beads with a buffer of your choice first.
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