Hi everyone,
I am trying to set up an experiment with the aim to identify putative transcription factors recruited in a functional regulatory region located in a non coding portion of human genome. ChIP-qPCR analysis are suggesting that the region has an enhancer activity and I am interesting to understand how this enhancer acts. Since I don't have information about putative transcription factors binding the locus I was wondering which would be my perfect positive or negative control for the pull-down assessment. Do you have any suggestion?
This is not an easy approach. Using your enhancer sequence to pull down nuclear DNA binding proteins can work if you, 1-remove non-specific DNA binding proteins which are very abundant, such as Ku70, 2- start with enough nuclear protein lysate so that you can purify nanogram amounts of your DNA binding protein for mass spec. Ideally, if you had a mutant enhancer that does not bind your factor or work as an enhancer to use as a control that could be used to remove non-specific binders.
- Badges
- Science topic