Entering edit mode
5.0 years ago
mkh
▴
60
Hi all,
I have a computation background and I have no idea about the experiment. From the experimental view, I would like to know how could I generate/design specific antibody/ies for a new Protein in order to do ChiP-Seq assay? Any reference, book or papers appreciated. I know this is a very general question and I do apologize in advance from all, too. Thanks,
do you have access to this book: https://www.springer.com/us/book/9781493973798 ? That should give you a good introduction into many aspects of ChIP-seq. In short, ChIP-seq grade antibodies are fairly tricky to produce; ideally you would find a company that has already used it.
For some proteins there are simply no good antibodies available. We have a project running in which we had to engineer the protein to have a so-called AviTag, an artificial appendix on the protein against which very good antibodies are available. This of course might alter the natural behaviour of the protein but is our only chance to ChIP this one in order to get its genomic binding positions. Examples for transcription factors with poor antibody availability are the ETS family transcription factors as well as several Jun factors.
Many thanks @Friederike and @ATpoint.
@Atpoint, how do you test if tag perturbing the binding of your target transcription factor or not?
You will never know for sure, but you can do general tests, e.g. whether the tagged protein still translocates to the nucleus and whether it's still able to bind DNA/chromatin in in vitro assays.
I know it is so too many questions but I could not find a place to ask my inquiries. I have seen in some papers the people raising target antibody in the rabbit and they use it for ChIP-seq assay. I am wondering how they modify that antibody and pull it down during ChIP process?
It seems that you will need to read up on the basics of antibody generation, e.g. here, here, here
For ChIP-seq, you would typically want a monoclonal antibody, and it is the antibody that one is using to pull down the DNA-protein complexes of interest. Once that immunoprecipitation step is done, the antibody is essentially discarded from the samples.
The main reason why antibodies are often raised in rabbits is that these are animals that meet some optimal threshold between "large enough to generate lots of antibodies at once" vs. "not too large to keep them in labs".
Good question. I will ask the colleague who works on the project what she has done and them come back to you.
Thanks! I appreciate it!