Immunoprecipitation

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:

• Co-Immunoprecipitation (Co-IP)
• Chromatin Immunoprecipitation (ChIP)
• RNA Immunoprecipitation (RIP)

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.

 

Basic Steps of IP

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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.

 

Experimental Considerations for IP

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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.

Considerations for Choice of Primary Antibody
Polyclonal Antibodies	Monoclonal Antibodies
Binds multiple epitopes on target protein.	Binds a single epitope on target protein.
Generally forms tighter complexes than monoclonal antibodies. Stable oligomeric complexes are formed since the antibody-antigen reaction is multivalent.	High-affinity antibodies may be preferred as low affinity antibodies may not form antigen-antibody complexes in solution.
Background may be high if some of the antibody has a low specificity and/or binds nonspecifically.	Background is low as the antibody recognizes a single antigen.
Even if the affinity of an antibody is low, oligomeric antigen-antibody complexes form easily due to multivalent binding.	Even if the binding affinity is low, several high-specificity monoclonal antibodies can be used in tandem to allow for multivalent binding.
An excess of the polyclonal antibody relative to the antigen may prevent the formation of oligomeric complexes.	An excess of the monoclonal antibody over the secondary antibody may result in a competition for the antigen, and end in a lower recovery.

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.

Resources:	Assaywise Letters:
Biological Detergents	Monitor Protease Contamination with Amplite™ Fluorimetric Assay Kits

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.

Considerations for Choice of Beads
Agarose	Magnetic	Agarose-Magnetic
Porous, mesh structure. Antibodies can diffuse and bind in the inner bead matrix, thereby providing high binding capacity.	Simple smooth spheres offer easy handling, short processing time, but a lower binding capacity.	Protein binding is comparable to agarose beads, and better than magnetic beads.
Typically ~100 μm, though size can vary.	~1.6 μm in diameter and can be very large.	~50 μm though can be larger.
Binding capacity is excellent, and the best compared to other methods.	Binding capacity is moderate and may not be high enough for some applications.	Like agarose beads, they have a high binding affinity to a resin matrix.
Sample loss may occur during washing steps.	Sample loss is negligible as pelleting only requires a magnet.
Centrifugation is necessary, though a magnetic rack is not.	Centrifugation is unnecessary though a magnetic rack is required.
Low reproducibility.	Highly reproducible results.	Medium reproducibility.
Provide poor, limited visibility of the beads upon precipitation.	Offer excellent visibility of the beads, and beads are easy to disperse after visualization.
Most inexpensive option.	Mid-priced option.	Most costly option.
Incapable of automation	Capable of high-throughput automation.

Product Ordering Information

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References

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