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Immunoprecipitation

It is possible to use antibody-antigen precipitation (immuoprecipitation) to isolate a specific antigen from complex protein mixtures such as cell or tissue lysates. Immunoprecipitation has proven to be an invaluable investigational tool that is routinely employed by many laboratories to ascertain critical information regarding a given antigen. These include small scale antigen purification for functional studies, N-terminal sequence analysis, investigation of protein-protein interactions, and the determination of the relative abundance and stoichiometric distribution of the antigen within a cell or tissue, among other things.

Success in an immunoprecipitation assay is dependent on two main factors, the abundance of antigen in the original sample, and the affinity of the antibody for the antigen (normally requires affinities of 108 mol–1 or higher)

Immunoprecipitation is Divided into Six Basic Steps
  1. Labeling the antigen (optional)
  2. Sample preparation: Lysis of cells to release the antigen
  3. Formation of the antibody-antigen (immune) complex
  4. Precipitation of the antibody-antigen complexes
  5. Analysis
  6. Troubleshooting
Before beginning the immunoprecipitation procedure, ensure that proper experimental controls are in place. Control antibodies should be as similar in nature to the specific antibody as possible. For a polyclonal antiserum, the ideal antibody negative control would be pre-immune serum from the same animal used for immunization. However, an equal concentration of non-immune (normal) serum from a different animal of the same species should suffice in its absence.
Antigen Labeling (Optional)
Antigen may be labeled by incubating in a medium containing a radioactive precursor, such as 3H-Thymidine, by iodination or biotinylation of surface proteins, by treatment with radioactive sodium borohydride, or by other published techniques.

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Sample Preparation
Prior to formation of antigen-antibody complexes, the antigen must first be efficiently extracted from the cell or tissue sample in a form that is still recognizable by the antibody. This requires that one take into account the location of the antigen within the cell (nuclear, cytosolic, membrane bound, etc.) and determine how best to extract the antigen with minimal effect on its structural integrity.

Before initiating an extraction/ cell lysis protocol one should consider what information one wishes to obtain from performing an immunoprecipitation. For example, is it necessary to extract functional protein for subsequent functional studies?

Additionally, one may want to consider whether they would like to disrupt protein-protein interactions or co-immunoprecipitate proteins that may interact with the target antigen within the cell. Based on these and other considerations, one must decide upon an appropriate lysis strategy. Often times an effective lysis strategy is determined empirically.

Perhaps the most important aspect of the lysis procedure is the composition of the lysis buffer. The ionic strength (salt concentration), choice of detergent and pH of the lysis buffer may significantly affect the efficiency of extraction and integrity of the antigen. Slightly alkaline pH and low ionic strength buffers typically favor protein solubilization while high salt concentration and low pH may cause the antigen to become denatured and precipitate from solution. The choice of detergents is crucial and may be influenced by many factors including (among others) the subcellular location of the antigen and whether one would like to preserve subunit associations and other protein-protein interactions.

In general, nonionic detergent (e.g., Triton® X-100, NP40) or zwitterionic (e.g., CHAPS) detergents tend to preserve non-covalent protein-protein interactions while ionic detergents (e.g., SDS, sodium deoxycholate) tend to be more denaturing of protein- protein interactions and may adversely affect the ability of the antibody to recognize the target antigen.

For a review of the characteristics of some commercially available detergents, see Appendix. Regardless of the lysis strategy employed, the lysis buffer should always contain a redundant cocktail of protease inhibitors to protect against proteolysis of the target antigen by proteases liberated during the lysis procedure, see appendix for protease inhibitors.



Additionally, all solutions should be pre-chilled and all steps in the lysis procedure should be performed on ice. The number of cells required for lysis will vary with cell line and the anticipated abundance of the target antigen in the sample. In general, lysates should be prepared from no less than 107 cells per mL of lysis buffer.

Following preparation of the lysate, determine protein concentration and adjust protein concentration of lysate to between 2 to 5 mg/mL with lysis buffer or PBS. Note that if Triton® X-100 was used in the lysis buffer, the protein concentration cannot be determined by absorbance of the solution at 280 nm as Triton® X-100 absorbs strongly at 280 nm. Extracts that will not be used immediately should be aliquotted and stored at -70̊C for future use. If the protein concentration of the extract is below 0.1 mg/mL, high quality BSA should be added to 1% (w/v) prior to freezing.

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Precipitation of Immune Complexes
To precipitate immune complexes, one may use Protein-A or Protein-G agarose, precipitating secondary antibodies, or Protein A-bearing S. aureus cells. The affinity of an antibody for Protein A or G is dependant on the subclass of the immunoglobulin and the species from which it came from. For example, Protein A is exceptionally well suited for immunoprecipitation of all rabbit primary antibodies, but not for chicken antibodies (see appendix for Protein A/G binding affinities).

To use Protein for immunoprecipitation of mouse primary antibodies, it is advisable to add 5 μg of rabbit anti-mouse IgG (secondary precipitating antibody) prior to the addition of Protein A/G, (mix gently, and incubate for an additional 30 minutes at 4°C prior to adding Protein A/G). When Protein A or G agarose is used for precipitation, a quantity of 10-20 μL of a 50% Protein A/G agarose slurry should be sufficient to precipitate the quantity of antibody/antigen complex prepared according to the procedure above.

Following addition of Protein A/G agarose, incubate with gentle agitation for 30 minutes at 4°C, then wash three times (or more, if the antigen has been radiolabeled) by centrifugation and resuspension in immunoprecipitation buffer, and collect antibody-antigen-Protein A/G complexes by centrifugation.

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Analysis
Pellets to be used for electrophoresis may be resuspended with 2x SDS sample buffer and resolved by SDS-PAGE for subsequent detection by Western blot. The purified immune complex may also be used for enzymatic studies, ligand binding, further immunizations, or other immunochemical techniques. These methods, when used in conjunction with immunoprecipitation, can greatly increase the amount of information discovered about an antigen.

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