Technique / Proteomics and protein biochemistry / Immunoprecipitation


Immunoprecipitation and co-immunoprecipitation



Immunoprecipitation and co-immunoprecipitation

Immunoprecipitation technique was first developed in 1974 to resolve the immunoprecipitated proteins on slab gels [1] and has since then become one of the most important techniques to study protein-protein interactions.

Two major factors affect the success of immunoprecipitation:

  • the abundance of the antigen to be studied
  • the affinity of the antibody used to study the antigene.

    Above factors are important for experiments that very rare acellular proteins are to be studied. With proper protein extraction and controls, even very low abundant proteins can be detected as a band in SDS-polyacrylamide gel.



    Immunoprecipitation image

    1. Antibody selection for immunoprecipitation

  • Polyclonal antibody: Excellent signal (hight avidity, can bind to multiple sites on the antigen); has some nonspecific backgrounds (by titrating the amount of antisera and more stringent washing, normally the background can be lowered)
  • Monoclonal antibody: high specificity for antigen; Signal depends on affinity of antibody; has some cross-reactions
  • Pooled monoclonal antibodies: Excellent signal; low cross-reactions, multivalent interactions.


    2. Immunoprecipitation procedures:

    a. protein lysate preparation. Tissue and cells can be lysed by different lysis buffer containing mild to strong detergent. RIPA buffer and NP-40 buffer are two most commonly used lysis buffer. Preliminary studies should be carried out to identify efficient lysis conditions, which mainly determined by salt concentration and detergent concentration. Most commonly used detergents are NP-40, Triton-X100, SDS etc. Precaution should also be taken to avoid protein degradation. Some commonly used protease inhibitors can be added to the lysis buffer:
    Aprotinin (1ug/ml)
    Leupeptin (1ug/ml)
    Pepstatin (1ug/ml)
    PMSF (50 ug/ml)
    Commercially available protein inhibitor cocktails are also available from vendors like Sigma and ThermoFisher scientific.
    To check the lysis efficiency, one can test by immunoblotting using the protein lysate and remaining cell pellet.
    For bacterial lysates, physical shearing by ultrsounic treatment is normally used for large quantity and cost efficient prepration.
    b. protein lysate preclearing (remove nonspecific background by pretreating lysate with a nonspecific antibody that will not interact with the chosen antigen).
    c. immune complexes formation by adding specific antibody, immune complexes extraction and purification.
    d. detection of enriched immune complexes by different methods, including SDS-PAGE electrophoresis, gel staining and western blotting.

    3. General protocol for immunoprecipitation

    - Wash cells in PBS.
    - Lyse the cells
    - Preclearing the lysate
    - Purifying the immune complexes
    - Downstream procedure for protein characterizations:
    - Determining half-life of protein antigen (puls-chase metabolic labeling)
    - Protein antigen steady-state expression level.
    - Relative molecular weight of the protein antigen
    - Post-translational protein modifications
    - Protein-protein interaction studies
    - Enzymatic activity detections

    Immunoprecipitation protocol (Yu Lab)
    Immunoprecipitation protocol for transfected cells (doc)
    Immunoprecipitation protocol (adapted from JBC publication)


    Reference:
    1. Horvitz H.R. 1974. J Mol Biol 90:727-38.

    Last update 15-Dec-2008, Rating of 1 votes.

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    By dotbio on 15-Dec-2008
    Immunoprecipitation application can be applied to 1) amplify the detection sensitivity of protein using immunoprecipitation. 2) determine the molecular weight of the unknown protein. 3) analyze the post-translational modification of the protein. 4) study the protein-protein interaction through co-immunoprecipitation technique. 5) useful for enzymatic activity detection when combined with native gel electrophoresis.
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