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  • Denatured or native protein in ChIP

    I'm a bit confused about the effect of formaldehyde crosslinking on protein conformation (in ChIPseq experiments).

    Do I understand it correctly that formaldehyde only partially denatures the proteins?

    Is there any information on how resistant is such DNA-protein cross-linked state to further changes in protein conformation?

    In example would the addition of EDTA to a zinc-finger protein open the DNA binding pocket and release the chromatin fragment?

    What about SDS? I have heard that crosslinked DNA-protein is resistant to SDS, yet others seem to claim that SDS increases the availability of specific protein areas to antibodies in subsequent bead-binding. If so, then wouldn't SDS also alter DNA binding stability?

    Looking forward to your input!

    Thanks

  • #2
    whatever is crosslinked remains covalently attached (until physico-chemical reversal). if your protein is fixed to DNA a conformational change will not disrupt the DNA interaction. Accordingly, the addition of EDTA will not release the DNA if the DNA has been crosslinked before and your crosslinker is not sensitive to EDTA.

    even though the crosslink can potentially lead to partial unfolding of the protein I would not call crosslinking a denaturing procedure in the first place. I guess that's too much of a stochastic event.

    In fact many ChIP protocols have SDS included in the IP. This will (partially) denature the protein and may help exposing epitopes for antibody binding. However, SDS does not affect the crosslink.

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