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Identifying a protein binding site in a particular gene's promoter region
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,
She indicates that her method for obtaining sequence information was to:
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
while her 10 bp EMSA consensus sequence is
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!
Re: Identifying a protein binding site in a particular gene's promoter region
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.
Last edited by admin; 09-14-2011 at 01:21 AM.
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