nuclear DNA binding

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nuclear DNA binding

Post by Balls » Tue Jan 22, 2013 9:03 am


I am wondering how I can demonstrate the association of a protein of interest with nuclear DNA, if nothing is known about the binding preferences at all.
Is there a standard that I might not be aware of?
I thought that preparing nuclear extracts, performing IP against the protein (many antibodies are available) and checking DNA levels in the pull-downed material against negative (matrix protein) and positive control (like Creb) would be a good idea... But there is also DNA in the cytoplasm and I want to exclude cytoplasmic binding elegantly.

Would be interested to know what you guys think...

Thanks for any response!

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Re: nuclear DNA binding

Post by jonmoulton » Tue Jan 22, 2013 6:39 pm

Sequence the DNA you pull down and check against the chromosomal sequence. It's useful to know where the protein is interacting with the DNA. That doesn't eliminate the possibility that the protein also binds cytoplasmic DNA, but if there is nuclear DNA binding you can nail that down.

You could also fluorescent-tag the protein and microinject it or otherwise transfect it into the cytoplasm, then watch where the signal goes. It would be interesting to see if it either accumulates in the nucleus or is excluded from the nucleus.

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