I have recently tried crosslinking mES cells with DSG at 2mM in PBS for 45 min followed by 15 min cross-linking with formaldehyde. However, I couldn't see any difference in the ChIP enrichment for my target proteins compared to cross-linking with formaldehyde only. Also, the chromatin sonication showed similar fragments size when (DSG+Formaldhyde) and (formaldehyde only) samples were sonicated under the same conditions.
Does that mean the DSG crosslinking did not work? is there any target protein where it is known to show a huge difference in enrichment using the dual cross-linking to be used as a positive control for my DSG cross-linking step?
Many thanks
Rajaei
Hi Rajaei,
For double cross-linking, I usually use the ChIP CrossLink GOLD (Diagenode) for 30 minutes and then proceed with a 10 minute crosslink with formaldehyde.
In my hands, in the case of proteins that are "easy to ChIP", the double crosslinking doesn't really affect the signal (it even sometimes raises the background level). However, I found it very useful for proteins that are more tricky to ChIP. In this case it worked fairly nicely.
Hope it helps,
Cheers,
Paul
Hi Paul,
Many thanks!! This seems to be the case in my experiment. I see a slightly increased background level but not the signal.
Cheers,
Rajaei
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