5 replies to this topic

[Mad Researcher]

Hi,

I am trying to understand what is a reverse coIP. I was searching on the internet, but with no luck yet. Could someone please explain what is a reverse coIP and when should it be done?

[bob1]

It's just looking for the reverse interaction in your IP. Your normal IP will look like this: IP protein A, then probe for protein B. The reverse IP of this will be IP protein B, probe for protein A. 

[Mad Researcher]

It's just looking for the reverse interaction in your IP. Your normal IP will look like this: IP protein A, then probe for protein B. The reverse IP of this will be IP protein B, probe for protein A. 

So basically, it's just the same protocol? I just interchange the proteins to see the interaction? 

[bob1]

Yes, you may have to play with the IP conditions a bit, depending on antibody, but the conditions should work out to be very similar to each other.

[Mad Researcher]

Yes, you may have to play with the IP conditions a bit, depending on antibody, but the conditions should work out to be very similar to each other.

This might sound stupid, but when I see an interaction between protein A and B, won't it be the same for B and A?

[bob1]

Yes it should be. However when you do an IP you won't just be pulling down the stuff bound to the antibody, there are also non-specific interactions with the antibody and the beads, which could be seen as a co-IP of A with B. The reverse co-IP sets out to confirm the interaction. In this case the antibody has a much much higher affinity for the protein B than the non-specific interaction with the beads, so you should then be able to see protein A precipitating with B.

 

Of course this relies on you having validated the antibodies first. Remember that western blots are denaturing and so not all antibodies that work for western will work with native forms of the protein. Ideally you see a single band with these antibodies on a western blot (preferably native gels too, but there can be isoforms on native gels, so multiple bands can be OK). You should also test that you can competitively prevent the binding by preincubation of the antibody with purified protein or peptide against the antibody epitope(s).