Immunoprecipitation (IP) is a small-scale, simple, rapid, affinity purification technique to enrich or isolate a protein from biological samples, like homogenized tissue or cell lysates. In IP, a specific antigen selectively binds with high affinity to an antigen-specific antibody, followed by the purification of the antigen-antibody complex by use of a solid-phase matrix, commonly Protein A or G agarose. IP is often used to identify and determine the relative abundance of interacting proteins.

Common subcategories of immunoprecipitation include:

Though other experimental methods can do the same, these methods rely heavily on a number of factors. Immunoblotting or western blotting (WB), for example, both rely not only on the molecular weight of the target protein, but also the rate of protein synthesis, degradation, and the state of target-specific post translational modification. For these reasons, IP is an extensively useful tool for isolating proteins and other molecules of interest, especially in the field of epigenetics to help determine protein structure, expression, identity, or extent of post-translational modification.

IP has found many uses as a preliminary assay, and can be coupled with a variety of in vivo or in vitro immunoassays, ELISAs, or immunoblotting experiments. Additionally IP techniques can utilize tags to identify unknown proteins and other components of a complex sample.

In a typical experimental setup, an antibody is first incubated with the cell sample containing the target antigen which leads to the formation of an antibody-protein complex. The complex is added to a matrix made of a protein coupled to magnetic, agarose (similarly, sepharose), or magnetic-agarose beads. The chosen protein has a high affinity for immunoglobulin (primarily, IgG), while the beads provide solid support for immobilization of the complex.

Next, the reaction undergoes a number of incubation and centrifugation steps whose purpose is to, firstly, join the antigen-protein complex and, secondly, remove the unnecessary supernatant. After the supernatant is removed, wash steps release unbound complexes, antibodies, or other undesired components and contaminants.

After, the protein complex is eluted, providing a purified antigen or protein as a final product. Finally, samples can be analyzed, traditionally through SDS-PAGE followed by staining techniques, WB, or mass spectrometry.

Though IP is regarded as a relatively simple technique, there still exist a range of factors that can impact the data accuracy of the experiment. In preparing the cell lysate, an appropriate lysis buffer must be chosen. The lysis buffer has a number of purposes and must stabilize the native protein conformation, minimizes antibody binding, site denaturation, inhibit enzymatic activity, all while increasing the amount of protein released from cells. Denaturing buffers containing NP-40 and Triton X-100 are often chosen, and phosphatase and/or protease inhibitors should always be added to the lysis buffer to prevent dephosphorylation and proteolysis of samples.

Primary antibodies must also be carefully considered (ideally, empirically determined) and antibody concentrations should be optimized through testing different ratios. Some considerations between using polyclonal versus monoclonal antibodies as a primary antibody are listed in the table below.

Another consideration in any IP experiment should be cell lysate preclearing through use of detergents. Pre-clearing potentially reactive components from a lysate before the IP experiment effectively prevents non-specific binding of lipids, proteins, carbohydrates, and nucleic acids to the resin matrix. Such nonspecific binding may adversely affect detection of IP targets. An appropriate concentration of salt and non-ionic detergent is commonly used to reduce non-specific binding so detergent optimization, beforehand, is key to a successful experiment.

It is also important to realize that the target protein and antibody are subject to degradation by proteases in certain samples. If specific proteases in samples are known or predicted, specific protease inhibitors should be used to prevent proteolytic degradation. When proteases are unknown, a combination of multiple small molecule inhibitors, like PMSF and EDTA, may be used.

Beads used for immobilization should be another major consideration for any IP experiment. Beads can be agarose, magnetic, or a blend of both. Some considerations for each of these types of beads are listed in the table below.

Immunoprecipitation: Applications, Uses, & Types
Immunoprecipitation
The principle and method of immunoprecipitation (IP)
Immunoprecipitation
Co-immunoprecipitation from Transfected Cells
Chromatin immunoprecipitation assay
RIP: RNA Immunoprecipitation