Question: Can A Mixture Of Antibody Be Used For Chip-Seq
2
gravatar for User 9088
8.5 years ago by
User 908820
United States
User 908820 wrote:

Hi,

If we want to study interaction of two proteins in transcription, can we use mixture of antibodies in ChIP-seq experiment? Is there any references that I can read?

Thanks,

ADD COMMENTlink modified 8.3 years ago by Chris Evelo10.0k • written 8.5 years ago by User 908820
2

Is there a bioinformatics/computational biology aspect to this question? If not, it would be better to ask in a molecular biology forum.

ADD REPLYlink written 8.5 years ago by Neilfws48k
2
gravatar for Lars Juhl Jensen
8.5 years ago by
Copenhagen, Denmark
Lars Juhl Jensen11k wrote:

I do not see a computational aspect of this question, so you may be better off asking elsewhere. That being said, I cannot see how using a mixture of antibodies would allow you to study the interaction of two proteins in transcription. If you have two antibodies that each target a different transcription factors, your ChIP would pull down DNA fragments bound by either of the two transcription factors. It would to my knowledge not give you any information on which transcription factor(s) bound which fragment.

If you want to understand to which extent the two transcription factors target the same set of downstream genes, I think they way to go is to do separate ChIP-seq experiments for the two transcription factors and then subsequently computationally analyze which promotors are bound by both transcription factors.

ADD COMMENTlink written 8.5 years ago by Lars Juhl Jensen11k
1

Again, there is no computational aspect to this, but some people do sequential IP to get at protein interactions, see e g http://nar.oxfordjournals.org/content/32/19/e151.full

ADD REPLYlink written 8.5 years ago by Mikael Huss4.6k
0
gravatar for Chris Evelo
8.5 years ago by
Chris Evelo10.0k
Maastricht, The Netherlands
Chris Evelo10.0k wrote:

In principle you can. There is no specific reason why the mixture of antibodies would not work. And since you will eventually access the precipitated sequences themselves you might be able to make an educated guess which precipitated sequence comes from what transcription factor binding.

In ChIP-on-chip this is not unheard of. Since the chips are expensive to run you will want to maximize the amount of information you get from a single array. The trade off is likely different for ChIP-seq.

There may be specific reasons to combine antibodies though. Suppose it is really a complex you are looking at, and the binding of protein A is conditional for the binding of protein B, then you will only get signals for both TFs combined. This is something you might in fact want to study using 2 antibodies. Although you will still have to evaluate the binding sequences for each of them, you might even find that protein B doesn't have a binding sequence of it's own but only piggy backs on protein A.

ADD COMMENTlink written 8.5 years ago by Chris Evelo10.0k

I agree that you can certainly use mixtures. However, I don't see how you would based on that study the interaction between TFs. But then again, I'm not an expert on the topic.

ADD REPLYlink written 8.5 years ago by Lars Juhl Jensen11k

I think you are right, the 2 antibodies alone are not sufficient for problems like this. You will want to combine that with other wetlab approaches, like using labeled anti-antibodies for detection of the complexes after separating those on a 2D gel. There might be smarter ways yo do the same. In my example knockout of protein A should remove any ChIP effects for both A and B.

ADD REPLYlink written 8.5 years ago by Chris Evelo10.0k

I think you are right, the 2 antibodies alone are not sufficient for problems like this. You will want to combine that with other wetlab approaches, like using labeled anti-antibodies for detection of the complexes after separating those on a 2D gel. There might be smarter ways to do the same. In my example knockout of protein A should remove any ChIP effects for both A and B

ADD REPLYlink written 8.5 years ago by Chris Evelo10.0k

But here is a real life example. Suppose the second TF is not really a TF but only complexes with the first. Then you will only see binding of the second antibody when you also see binding of the first, but not the other way around. Also you will only see effects on expression when both antibodies bind and thus the TF was bound and complexed. Or sometimes opposite effects if the complex is formed, from what you would see with the first antibody alone.

ADD REPLYlink written 8.3 years ago by Chris Evelo10.0k
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