In Co-IP, complexes of two (or more) proteins are isolated using a procedure similar to the IP procedure.
Co-IP is often used for the analysis of interactions of multiple proteins and their functions.
The principle of Co-IP is the same as IP, except that the proteins associated with the antigen are also precipitated.
A protein complex is isolated by Co-IP using an antibody for one of the components in the complex.
The choice of antibody is critical for successful Co-IP. The antibody must bind to the surface of the complex. Alternatively, a component is tagged, and IP is performed using an anti-tag antibody. In either case, the method of cell lysis and the conditions of washing should be carefully evaluated because components in protein complexes are often held in place by weak interactions.
A common approach is to optimize the condition for Co-IP using a tagged protein, which is easy to detect, and then perform Co-IP experiments with endogenous proteins.
[Related topics] Pull-down assay experiments using Tagged protein purification kits
Complex formation identified by Co-IP should be confirmed by other methods, such as analysis of protein interactions in vivo using fluorescent-labeled proteins.
[Related topics] Protein-Protein Interaction analysis tool "Fluoppi"
[Related topics] Protein-Protein Interaction Detection System "CoralHue™ Fluo-chase Kit"
|Endogenous proteins||Tagged proteins (pull-down assay)|
|Main advantages||Protein complexes are isolated in a relatively natural state.||An N- or C-terminal tag is likely available for antibody binding after complex formation. Antibody binding is unlikely to interfere with complex formation.|
|Issues to consider||The epitope may be buried upon complex formation. Antibody binding may interfere with complex formation.||The expression levels of recombinant proteins are substantially higher than those of their endogenous counterparts, which may result in artifactual results.|
|Anti-tag antibodies suitable for IP||List of monoclonal anti-tag antibodies
List of polyclonal anti-tag antibodies
List of anti-tag antibody-coated agarose beads
List of anti-tag antibody-coated magnetic beads
List of anti-tag antibody-coated magnetic agarose beads
|Pull-down assay-related kits,
gels and elution peptides
|List of tagged protein purification kits
List of tagged protein purification gel and elution peptide sets
|Primary antibodies suitable for IP (excluding anti-tag antibodies)||List of monoclonal primary antibodies
List of polyclonal primary antibodies
List of antibody-coated agarose beads
List of antibody-coated magnetic beads
List of antibody-coated magnetic agarose beads
|Others||Protein G-coated magnetic beads
|For analysis of immunoprecipitated proteins||HRP-DirecT series
Co-eluted antibody is not detected with HRP-labeled antibodies in Western blotting of immunoprecipitated proteins. It is especially useful when the target protein band overlaps with the heavy or light chain band. Analysis time is also reduced.
|For confirmation of complex formation||
Protein-Protein Interaction analysis tool "Fluoppi"
Protein-Protein Interaction Detection System "CoralHue™ Fluo-chase Kit"