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Odd result help


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#1 Borrowing Time

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Posted 07 July 2011 - 08:29 AM

Hi,

I seem to be having an odd result and I'd like to know what you all think. I am new to this forum. Any help would be appreciated.

I have transfected a protein into my cell lines which appears to be significantly down-regulating my protein of interest. I have verified this down-regulation on a protein level thoroughly by Western blotting (on multiple cell lines, with multiple repeats.) This down-regulation is also on an mRNA level which I have shown by qPCR (on multiple cell lines, with multiple repeats.)

Now here comes the caveat. The protein I've transfected in is a transcription factor. So I was expecting to see a down-regulation of promoter activity in my protein of interest but when I cloned its promoter with the putative binding site for the TF, I actually observe an UP-regulation of Luciferase activity!!! It's odd and counter-intuitive and does not make sense. Help!

#2 pcrman

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Posted 08 July 2011 - 08:59 PM

Many things should be taken into consideration.

1) the cloned promoter may not truly recapitulate the endogenous promoter and that is why you get a different result
2) Reporter assay is very error prone, make sure you have stringent controls
3) You can do ChIP assay instead of reporter assay to pull down the transcription factor, and then to see if it is enriched on the predicted target site on the promoter of its regulated genes.




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