Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.
Immunoprecipitation (IP) is the small-scale affinity purification of antigens using a specific antibody and is one of the most widely used methods for antigen purification and detection.
This approach enables researchers to:
identify the activation status of proteins
determine post-translational protein modifications
measure the molecular weight of a given protein
capture protein-binding molecules in the study of protein-protein and protein-nucleic acid interactions
Target antigens are usually immunoprecipitated from complex solutions, such as cell lysates, the goal being to isolate and eventually detect and measure a specific protein (i.e., the antigen of the specific antibody). The basic protocol for performing an IP is diagrammed below, where the order (sequence) of steps can be done in two different ways.
In one sequence (left), an antibody (monoclonal or polyclonal) against a specific protein is pre-immobilized onto an insoluble support, such as agarose or magnetic beads, and then incubated with a cell lysate containing the target protein. During the incubation period, gentle agitation of the lysate allows the target antigen to bind to the immobilized antibody. The immobilized immune complexes are then collected from the lysate, eluted from the support and analyzed based on the nature of the target antigen.
Alternatively (right), free, nonbound antibody is allowed to form immune complexes in the lysate and then the complexes are retrieved by the beads. While the pre-immobilized antibody approach is more commonly used for IP, using free antibody to form immune complexes is beneficial if the target protein is present in low concentrations, the antibody has a weak binding affinity for the antigen or the binding kinetics of the antibody to the antigen are slow.