chIP assay - why crosslinking? (Jul/29/2009 )

I was wondering why to crosslink?

by crosslinking every protein will be bind to DNA. so what the point since we want only the protein of interest.
by crosslinking you force the protein to bind even they don't bind. So you end up with protein binding to your promoter but is it a real binding or the result of cross linking.

am i right?

ulujm on Jul 29 2009, 09:49 AM said:

Crosslinking is not total. Proteins in the vicinity of DNA will crosslink to the DNA, which would not be the case for most proteins. Also, specificity is achieved through repeated experimentation. A protein that just "happens" to be near your promoter region, but is not specific, will not likely be seen in repeated experiments. Also, as is the case with all experiments, results should be supported by other data as well to become meaningful.

so what about the reverse crosslink? it should unbind everything right?

or only a strong binding protein will stay?

You should only reverse crosslink an aliquot of your sample in order to quantify the DNA. You can then proceed with the cross linked sample to the IP stage.

This is crosslinking is not total. Proteins in the vicinity of DNA will crosslink to the DNA, which would not be the case for most proteins. Also, specificity is achieved through repeated experimentation. A protein that just "happens" to be near your promoter region, but is not specific, will not likely be seen in repeated experiments.

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