western blot after ChIP - necessary to revert crosslinking? (Jun/05/2006 )

hi,
is it necessary to revert crosslinking after ChIP just to do WB? i mean, will the protein enter the acrylamide gel with the DNA "sticked" to it?
if reversion of crosslinking is necessary, how can i do that? because i guess the protein will be degraded after 4 hours at 65ºC.
thanks a lot

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Hello,

It depends on wether your protein is involved in protein complexes since formaldehyde will also crosslink proteins. What I did and seemed to be enough to reverse crosslinks, at least for the protein I studied, was to boil it in the loading buffer for 10 min before loading on the gel.


Hope this helps.

Vero

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is boiling in the loading buffer for 10 min enough to reverse the crosslink?

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Hi,

I usually do a reverse crosslink before loading onto the gel. I do as follows:

- Add 4% of 5M NaCl to my sample
- Incubate at 65 degrees for 5 hours
- TCA precipitation.

And my proteins don't get degraded at all :-)

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You can just take some of your eluted sample and then boiled for 10 min after adding the loading buffer and 2-ME. It works well.

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thanks for your answers!
so, if reversal of the crosslink with NaCl is not necessary, i guess elution neither. i mean, won´t it be enough to add sample buffer to the immunoprecipitate?

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Yes, actually the 1×sample loading buffer is more violent than the elution buffer. You can just do it as CO-IP assay. But do not forget to add 2-ME and boiled the loading buffer eluted sample. Just try it. It works well in my hand.

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