I'm from the protein field, and I'm trying to gain more knowledge about genomics technologies. So this might be a very stupid question. I read that in CHIP-seq people use antibodies to detect the transcription factor of interest. However, we know that antibodies can give a lot of problems.
Is it not possible to use recombinant protein (TF, histones) to find the binding sites in DNA instead? Would this give lot of non-specific binding?
Hi Czuee,
I'm a little unclear on what you would like to do with the recombinant proteins.
Do you mean expressing a recombinant TF or histone carrying an epitope tag (e.g. GFP, FLAG, HA) to it? If you do this, you'll still need an antibody against the tag itself so won't get around using antibodies (though admittedly the antibodies against epitope tags are usually strong and well-validated).
There are some tags (e.g. Streptavidin) which can bind directly to ligands (e.g. biotin for streptavidin) and thus allow you to purify a tagged protein without use of an antibody.
However, keep in mind that, while adding an epitope tag to your protein may solve some problems, it can cause others - for example they may change the ability of your TF to bind to DNA or other interacting partners. Also, if you're looking at post-translational modifications such as epigenetic marks (since you mention histones), you obviously won't be able to selectively purify marked histones with an epitope tag, you'll just get the entire tagged-histone population
What sort of problems are you having with your antibody, out of interest? It may be easier to resolve those problems than developing new systems!
I hope that helps - do let me know if I've misunderstood your question.
Cheers,
Dan
Daniel, thanks for your quick reply.
I'm not looking for a solution to a problem but rather trying to develop a potential project for a postdoc in this field! This was just a random idea that came to my mind, and I thought I will ask the experts.
By using a recombinant protein, I meant expressing the TF in E.coli or another system, purifying it and then using it to bind to DNA. Now that I think about it, I realize that this will give all potential binding sites and not the ones bound under a certain condition. Also, the native proteins would need to be removed first.
Concerning modifying histones, I found this interesting paper where they have developed chemical techniques to modify histones. http://www.nature.com/nmeth/journal/v11/n8/full/nmeth.3022.html
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA