I want to test whether two proteins A and B could interact with each other, and I did Co-IP.
According to my result, I found that B-IP could detect A, but I can not detect protein B in anti-A-IP. If A and B can interact with each other, I should detect B in anti-A-IP.
I am sure that both A-IP and B-IP is successful.
So what could be the reasons that I could detect A in B-IP,but it did not work in the opposite way?
Many thanks.
Maybe the anti-"A" protein binds an epitope at or near the binding site for B, and thus disrupts the interaction either via steric interference or direct competitive binding. Alternatively, the antibody might act allosterically by changing the conformation of A such that it no longer binds B. Try another antibody directed against a different region of the A protein, instead.
The less favorable interpretation for your working hypothesis is that A and B do not interact and that you have a false positive in your anti-B directed co-IP. The two most important controls to rule this possibility our are 1) make sure your anti-B antibody does not cross-react with the A protein, and 2) perform your experiment in a B knock-out or B knock-down lysate, and demonstrate that A is no longer detected in the negative control IP.
In addition to Daniel Cohen’s advices, you also can try to perform GST-pull downs with your protein.
You may tag your “B” protein with GST, and incubate it with a cell homogenate containing the “A” protein. If you want to show that the interaction A-B is direct, you can use both purified proteins.
In addition, you can try moving the epitope from N to C terminus or vice versa.
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