De-crosslinking DNA-protein IPs

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Wed Oct 23 19:27:03 EST 2002


On Wed, 23 Oct 2002 15:24:46 -0500, "EK"
<khatipovNO-SPAM at NO-SPAMuchicago.edu> wrote:

>Hi everybody,
>A colleague of mine is struggling with the following question. Would boiling
>in standard SDS-PAGE loading buffer de-crosslink the proteins she fished our
>via IP with the Abs against one of the proteins in the crosslinked complex?
>She used a DNA-binding protein (nuclear) as a bait. Before IP, she
>crosslinked DNA and proteins with paraformaldehyde. There is a protocol she
>knows that says incubating at 65C overnight with 1%SDS and high salt will
>reverse crosslinking, but I am quite skeptical about that. Is this at all
>possible to reverse PFA crosslinking ?
>

I'm skeptical, too. Has she tried a reversible cross-linker? Check the
Pierce catalog; they have a bunch.

Nick

-- 
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu



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