IP / co-IP

Immunoprecipitation / co-Immunoprecipitation

Immunoprecipitation (IP), Co-immunoprecipitation (Co-IP) and Pull-down assays are approaches designed to study the presence, abundance, up regulation or down regulation, post translational modifications and interactions of proteins. Previously, the standard purification and detection methods were radio-immuno labeling assays (RIA). The use of radioactive isotopes to label antigens can still be found in use today, however, safety and regulatory concerns along with cost have led to the development of new methods free from this type of labeling. With advances in these new techniques, antigens are able to be purified directly from the lysate, resolved by SDS-PAGE and then detected by western blotting, ELISA or quantified by mass spectrometry with the equivalent sensitivity that was first seen in radio-immuno labeling assays.

Immunoprecipitation Assay
The Immunoprecipitation assay (IP) is designed to detect and purify a specific protein from a homogenate, such as cell or tissue lysate. This method involves incubating an antibody specific to the protein of interest along with cell or tissue lysate to form an immune complex. This complex is then precipitated onto an immobilized support, typically agarose resin. Any protein not bound to the agarose resin is subsequently removed during the washes. Finally, protein is eluted from the beaded support through a series of washes and collected through centrifugation. The sample is analyzed by SDS-PAGE, often followed by Western blot detection.

Immunoprecipitation Protocol (with reagents list)

Co-Immunoprecipitation Assay
The Co-Immunoprecipitation assay (Co-IP) is based on the same methodology as immunoprecipitation in its ability to capture and purify an antigen of interest; however, Co-IP is focused on the additional molecules that are bound to the target protein by inherent interactions in the sample complex. These interacting proteins may be complex partners, structural proteins, co factors, or signaling molecules. The significance of the Co-IP assay is useful its identification of protein to protein interactions thereby elucidating signaling pathways.

Co-IP is the most widely used in vitro method for protein-protein interaction discovery and verification of interactions seen in other systems such as the yeast two-hybrid system (in vivo method). This affinity-based molecular pull-down method, facilitated by epitope tagging of recombinant proteins, has enabled rapid and detailed studies of expression, function and interaction of proteins that may fuel discovery of new drug targets and therapeutic molecules.

Read more in the FLAG® 96-well Immunoprecipitation System application note.