Did you have protein B enzyme activity before co-IP?

Did you change pH to the optimal pH for measuring enzyme activity?

Are you sure A interacts with B? No cross-linking should be needed if A interacts with B.

In cell lysates, Protein B activity is present.

After elution, pH was neutralized that is also suitable for activity assay.

I am not 100% sure, of course, about Ap-Bp interaction but that is the hypothesis. I thought if the interaction is weak, I can break that interaction during cell lysate preparation etc.

Thanks for your help.

Apart from crosslinking try high salt that helps if interactions are more hydrophobic than hydrophilic

And try to dilute as little as possible as , you know, equilibirum depend on the concentration; if concentration of A or B is low there will be less A-B complexes.

Best of lucks

RF

I am not certain this will help, but consider modifying the following protocol to cross-link antibodies to proteinG-agarose beads (http://www.abcam.com/protocols/cross-linking-antibodies-to-beads-protocol). Even if it works, it probably isn't the "turn-the-key" solution you would have preferred.

I've used it in the past. The cross-linking and the subsequent IP both work. The fact that IP works indicates that the antibody isn't denatured.

Best of luck