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ChIP antibody question


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#1 Dani123

Dani123

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Posted 27 May 2010 - 01:15 AM

Dear colleagues,

I want to find binding sites of my transcription factor via ChIP-seq. I have a polyclonal antibody that weakly recognizes my protein in western (besides numerous other bands) and I am not sure if I can use that antibody for ChIP. How could I test that? I don't have any target candidates that I could use.

I am also thinking of tagging my protein in order to do the ChIP with a ChIP grade antibody against the Tag:
1. What is your experience with that?
2. Does overexpression of the tagged protein have negative influence on the ChIP assay? Should I use a weak promoter for that? Also the usage of a Tet inducible system would be possible; I would be able to titrate Dox for low expression levels.
3. Is a knockdown of the endogenous transcription factor neccessary?

Thanks a lot for you help!!!

Daniel




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