De-crosslinking DNA-protein IPs
hubahopp at gmx.de
Mon Nov 4 07:43:09 EST 2002
Crosslinking proteins with formaldehyde will yield imines in a chemical
equilibrium reaction. when you remove the crosslinker (as you do by
diluting with sample buffer), the equilibrium is shifted towards the
unbound compounds - the longer and warmer you incubate, the more.
You should be able to preserve the complexes by reducing them with sodium
cyanoborohydride, the amines produced are stable. Protocols on BH3CN use
should be at avail by a simple googling.
Another possible crosslinker would be glutaraldehyde which is bifunctional.
It also requires subsequent reduction.
At 15:24 23.10.02 -0500, EK wrote:
>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 ?
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