Question: Specific Antibodies for ChiP-Seq assay
0
gravatar for mkh
5 weeks ago by
mkh30
USA/Ann Arbor/Biological Chemistry
mkh30 wrote:

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,

chip-seq • 145 views
ADD COMMENTlink written 5 weeks ago by mkh30
1

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.

ADD REPLYlink written 5 weeks ago by Friederike4.2k
1

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.

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by ATpoint16k

Many thanks @Friederike and @ATpoint.

@Atpoint, how do you test if tag perturbing the binding of your target transcription factor or not?

ADD REPLYlink written 5 weeks ago by mkh30
1

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.

ADD REPLYlink written 5 weeks ago by Friederike4.2k

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?

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by mkh30

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".

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by Friederike4.2k

Good question. I will ask the colleague who works on the project what she has done and them come back to you.

ADD REPLYlink written 5 weeks ago by ATpoint16k

Thanks! I appreciate it!

ADD REPLYlink written 5 weeks ago by mkh30
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1141 users visited in the last hour