Co-immunoprecipitation (Co-IP)

Introduction
Co-immunoprecipitation, Co-IP in short, is a widely applied technique to identify physiologically-relevant protein-protein interactions by utilizing target protein-specific antibodies to indirectly capture proteins that are bound to this specific target protein. As an extension of IP, Co-IP can capture and purify not only the primary target, but also other macromolecules binding to the target by native interactions. Difference between IP and co-IP is the focus of the experiment. IP is focused on the primary target, which binds the antibody. Whereas, Co-IP targets the secondary targets, which interacts with the primary proteins, instead of antibody. Following immunoprecipitation, proteins trapped on the beads are washed and eluted to gain purified primary and secondary target proteins. These target proteins can be characterized in various methods, such as WesternBlot and mass spec analysis under shotgun strategy, to identify partner protein IDs, binding affinities, kinetics of binding and undiscovered functions of the primary protein.

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Critical factors about Co-IP:

As a powerful technique, Co-IP is utilized regularly by biochemists to explore protein–protein interactions. While the Co-IP methodology is straightforward, identifying physiological protein-protein interactions through Co-IP reaction is not easy, because of the instability of the interaction, nonspecific binding to IP reagents and antibody contamination. All of the problems above can have negative effects on detection of protein-protein interactions.

Co-IP of protein complexes

Since Co-IP depends on protein-protein interactions to detect the bound proteins, the binding affinity and stability of the physiological interactions during the whole Co-IP process is very important. Inadequate binding time and extensive washing steps may reduce the binding efficiency and cause a failure of detection of protein interaction. Therefore, for proteins, which are predicted to have week binding affinity or dynamic interactions, advance stabilization of protein-protein interactions are necessary, for instance, crosslinking processes can be performed before Co-IP.

Unspecific interactions

Breakage of cell membrane and organelles cause releasing of large of amount of proteins, which are separated by the boundary, to come into contact. Therefore, it is inevitable that unspecific interactions, in other words, false positive interactions, may occurs, which interfere with the data analysis. This is especially common for proteins that have unfolded or flexible regions, which are relatively sticky and unspecifically binds other proteins. Ways to relive such problems can be preclearance of lysate using primary antibodies to remove unspecific proteins, and changing the ionic strength of the buffer to reduce the unspecific binding.