No product after chIP? - (Jan/05/2007 )
I have spent several months trying to carry out chIPs on 2 cloesly related genes.
I have been able to demonstrate, quite nicely, the binding of 2 transcription factors to the promoter region of one of these genes but not the other. This is despite the fact that both promoter regions contain potential binding sites for these transcription factors. Also, using RNAi and pharmacological inhibitors to block these transcription factors, I have been able to down-regulate expression of both genes of interest.
Obviously the chIP assay is working as I can detect a PCR product following IP for the first gene of interest. And I can detect a PCR product for the second gene of interest in the input sample but not the IP, indicating the primers are OK. However, our other evidence would suggest that the transcription factors should bind to this gene.
Any suggestions?
I have been able to demonstrate, quite nicely, the binding of 2 transcription factors to the promoter region of one of these genes but not the other. This is despite the fact that both promoter regions contain potential binding sites for these transcription factors. Also, using RNAi and pharmacological inhibitors to block these transcription factors, I have been able to down-regulate expression of both genes of interest.
Obviously the chIP assay is working as I can detect a PCR product following IP for the first gene of interest. And I can detect a PCR product for the second gene of interest in the input sample but not the IP, indicating the primers are OK. However, our other evidence would suggest that the transcription factors should bind to this gene.
Any suggestions?
Maybe you should try an EMSA? Here's a hand-waving possibility: for your second gene, a third protein comes into play as part of a complex, and when you cross-link it with the two others on the promoter, you block the epitopes that you can detect when the two TFs are bound to the first gene's promoter?
I've no useful suggestions for ChIP as I'm here looking for help on it. Sigh.
