Immunoprecipitation
From Wikipedia, the free encyclopedia
Immunoprecipitation (IP) is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. This process can be used to enrich a given protein to some degree of purity or to concentrate a low-abundance protein. Co-immunoprecipitation can identify interacting proteins or protein complexes present in cell extracts.
By selectively targeting a known protein that is believed to be member of a larger complex of proteins, one can (in principle) pull the entire protein complex out of solution. This concept of pulling complexes of multiple interacting proteins out of solution simply by immunoprecipitating a single member of the protein complex is regularly referred to as a "pull-down". In actual practice, one does not expect to actually be able to pull-down and identify just all members of a complex in a single pulldown. A typical strategy would be to perform the pull-down, identify new members of the complex and then perform a new series of immunoprecipitations wherein the newly identified members are targeted for immunoprecipitation. This strategy will often result in the discovery of even more members of the complex as well as result in the rediscovery of the originally targeted protein, an important vaildation that you are working with the correct complex.
Protein complexes, once bound to the specific antibody, are removed from the bulk solution by capture with an antibody-binding protein attached to a solid support. Historically the solid support has been microscopic highly-porous agarose beads (slurries). More recently, monosized superparamagnetic beads have been available as a support material which offers certain unique advantages over the porous agarose beads such as the ability to bind larger complexes (all binding occurs on the bead surface), faster binding kinetics (protein complexes do not have to penetrate into a porous surface) as well as decreased background (fewer false positives).
There are two general strategies available to the actual immunoprecipitation. The antibodies which bind to protein to be "precipitated" can either be i) added directly to the solution containing the protein mixture or ii) incubated with the beads and allowed to bind to the beads followed by incubation with antibody bound beads with the solution of proteins. In the first scenario once the antibodies have been given sufficient time to bind to their target proteins, the beads are added to the solution and the antibodies will then adhere to the beads. In the second scenario, the beads with bound antibodies are added to the sample and incubated to allow binding of the protein or complexes of proteins to the immobilized antibodies. Either scenario gives the same end-result with the protein or protein complexes bound to the antibodies which themselves are immobilized on the beads of choice (be they sepharose agarose or monodisperse superparamagnetic). The antibody-binding proteins (Protein A, Protein G, Protein L) were initially isolated from bacteria and recognize a wide variety of antibodies, although each has its own specificity.
Following the initial capture of a protein or protein complex, the solid support is washed several times in an attempt to remove any proteins not specifically and tightly bound to the support through the antibody. After washing, the precipitated protein(s) are eluted and analyzed using gel electrophoresis, mass spectrometry, western blotting, or any number of other methods for identifying constituents in the complex. Thus, co-immunoprecipitation is a standard method to assess protein-protein interactions.
Immunoprecipitation is very useful in chromatin immunoprecipitation, which allows analysis of DNA sequences bound to histones or other chromatin components.
[edit] Steps
- Lyse cells and prepare sample for immunoprecipitation.
- Pre-clearing the sample using serum from the animal species in which the primary antibody was raised (this decreases background and non-specific proteins).
- Incubate solution with antibody against protein of interest (antibody can be attached to solid support before or after this step) and allow antibody-antigen complex formation.
- Precipitate the complex of interest, removing it from bulk solution.
- Wash precipitated complex several times. Spin each time between washes and remove supernatant. After final wash, remove as much supernatant as possible (this step is most efficient when performed using spin columns so the entire volume of buffer can be removed at each wash step).
- Elute proteins from solid support (usually with low-pH or SDS sample loading buffer).
- Analyze complexes or antigens of interest. This can be done in a variety of ways:
- SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); expose the film if you used radioactivity.
- Transfer and Western Blot using another antibody for proteins that were interacting with the antigen.
- Other molecular methods.
[edit] External links
|
|||||||||||
