Identifying a protein binding site in a particular gene's promoter region

[minimal]

Hello all,

I recently started working in a new lab. Some of my assignments include working on projects that were started by other people many years ago (in this case, a PhD candidate) who no longer work here and are unable to be contacted.

Our lab is collaborating with another lab that is performing CHiP on some of our samples. They wrote us saying,

I noticed that in the paper you indicated that you had already identified the protein subunit binding site in the gene promoter. Can you send me that sequence please?

The italicized items are specific, as in p10 and Her2 or something, but I feel that I shouldn't include them.

She indicates that her method for obtaining sequence information was to:

• Gene upstream sequence from ensembl.org
• Use P-Match to search 3000 bp upstream for putative protein binding sites
• Design primers targetting these regions using Primer 3
• Perform chromatin immunoprecipitation analysis

She then lists a table with the sequences of primers designed for one of four binding sites for CHiP.


However, I have gone back through her thesis, and she identifies a consensus binding site on the protein, that she used for EMSA, and it is totally different from the sequences that she identifies for CHiP. She states

The following table shows the sequences of the target probes that are DNA fragments containing the protein subunit binding consensus sequence. These 20 bp sequences were designed so that they included the 10 bp consensus binding sequences detected on the fourth binding site (the site closest to transcription start site

Fine, great, but that sequence that she then prints is totally different. For instance let's say the fourth binding site for ChiP is
AAAGTCGCGCAG
while her 10 bp EMSA consensus sequence is
CAGTCCCCTT

Finally, when I go to sabiosciences, and do a qPCR CHiP primer search for mouse combining the protein and gene, I don't see *any* of the binding sites that she indicated. Are they not ubiquitous? Would there be a difference between ensembl and sabiosciences?
Does anyone know why her two are different? Moreover, why do her two not match any of the ones listed by sabiosciences?
In your opinion, which one of hers should I give him?

I imagine there will likely need to be followup questions answered to this.

If you made it through all of this, thanks.
If you made it through all of this and manage to reply, thank you very much!

[admin]

Makes sense as ChiP is a molecular biology technique whereas a protein binding site on DNA would not be dependent on Chromatin Immunoprecipitation (which depends on Antibody - Chromatin - DNA recognition).

Having done many promoter analysis and EMSA myself, I personally would respect the protein binding site data (ie Matinspector and promoter analysis data - especially if confirmed experimentally - much more than ChiP sites)

The protein-DNA binding sites on the promoter should be found in the databases, however ChiP primers may vary depending on how they were created/obtained.

[minimal]

Thanks!