how to check Cross-linking efficiency? - (Nov/08/2008 )

Hi everyone, I'm just starting to trying out some chIP protocols for plants. Looking at several protocol, none of them gave me any clues on how to check the efficiency for cross-linking. I sort of have some clue, does it have to do with using reverse cross-linking and checking to see if the reversed sample are shifted less than non-reversed sample on an agarose gel?

Any help would be greatly appreciated:)

Also is there need to do a titration of cross-linking %? What defines a good cross-linking vs a bad one?

Marc

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Everyone that I've talked to that does ChIP assays uses the assay itself as a readout of crosslinking efficiency (i.e. if the assay worked then the crosslinking worked). The readout for the assay (PCR) is much faster, in my opinion, than trying to do Southern or western blots. Also, if you look around at protocols for ChIP you won't find a great deal of variation in crosslinking times and formaldehyde concentrations since there is a limit to the amount of crosslinking you can do and still get efficient shearing of your DNA (at least if you are using a sonicator). And if you are looking at histones and histone modifications, crosslinking may be of limited benefit, since they stay bound to the DNA with or without the crosslinking.

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So I guess the bulk of the check somes from sonication step right? What are the controls needed for sonication?
For example,
sonicated sample at different speed/condition, pre sonicated sample, pre sonication without lysis, pre sonication without cross-linking?

Thank you
Marc

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To check that the crosslinking worked and check the shearing efficiency at the same time you could do the following:
Sonicate crosslinked and non-crosslinked samples. Divide samples in half and reverse crosslinks and protienase K treat one of the halves. Run the samples on 1% agarose. For the samples that weren't crosslinked there should not be any difference if the reversal of crosslinking procedure was run or not (i.e. both lanes should have smears at relatively low MW). If the samples were crosslinked though, only the ones with crosslinks reversed should enter the gel and give you a smear smaller than 10kb (the ones without crosslink reversal should still be stuck in the well with a bit of very high MW smear). If there's no difference with or without crosslink reversal then the crosslinking probably didn't work (though I would find this unlikely). If the crosslinking worked, then you can check the shearing efficiency by determining the average fragment size in the lane with the crosslinked sample that had crosslinks reveversed.

I could probably state that in a much easier way if I had a little more sleep but hopefully you get the point.

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Thank you KPDE, yea I think i'm gonna take that approach as well.

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Thank you KPDE, I think i'm gonna take this approach.

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