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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. Co-immunoprecipitation (also known as a 'pull-down') can identify protein complexes present in cell extracts: by IPing one protein believed to be in a complex, additional members of the complex can also be identified. The complexes are brought out of solution by insoluble antibody-binding proteins isolated initially from bacteria, such as Protein A and Protein G. These can also be coupled to sepharose beads that can easily be isolated out of solution. After washing, the precipitate can be analyzed using mass spectrometry, western blotting, or any number of other methods for identifying constituents in the complex.

Immunoprecipitation is very useful in chromatin immunoprecipitation, which allows analysis of DNA sequences bound to histones or other chromatin components.

[edit] Steps

1. Lyse cells and prepare sample for immunoprecipitation. (you can radiolabel cells to easily assay for your protein later)
2. Pre-clearing the sample using serum from the animal species in which the primary antibody was raised (this decreases background and non-specific proteins).
3. Incubate solution with antibody (usually poly-clonal against protein of interest) and allow antibody-antigen complex formation.
4. Precipitate the complex of interest using insoluble antibody proteins such as Protein A.
5. Analyze complexes or antigens of interest. This can be done in a variety of ways:
   1. SDS-PAGE electrophoresis (expose the film if you used radioactivity).
   2. Transfer and western blot using another antibody for proteins that were interacting with the antigen.
   3. Other molecular methods.

[edit] External links

• Analysis of Proteins Using Immunoprecipitation

Proteins: key methods of study

Experimental : Protein purification | Green fluorescent protein | Western blot | Protein immunostaining | Protein sequencing | Gel electrophoresis | Protein immunoprecipitation | Peptide mass fingerprinting Bioinformatics : Protein structure prediction | Protein-protein docking | Protein structural alignment | Protein ontology | Protein-protein interaction prediction Assay: Enzyme assay | Protein assay | Secretion assay

de:Immunopräzipitation

1. Lyse cells in ~500uL lysis buffer and prepare sample for immunoprecipitation. (you can radiolabel cells to easily assay for your protein later) 2. Pre-clearing the sample using serum from the animal species in which the primary antibody was raised (this decreases background and non-specific proteins).

 or: pre-clear the sample with ~20uL prtein A/ protein G beads.

3. Incubate solution with antibody (usually poly-clonal against protein of interest) and allow antibody-antigen complex formation. 4. Precipitate the complex of interest using insoluble antibody proteins such as Protein A. 5. Analyze complexes or antigens of interest. This can be done in a variety of ways:

    1)SDS-PAGE electrophoresis (expose the film if you used radioactivity). 
    2)Transfer and western blot using another anitbody for proteins that were interacting with the antigen. 
    3)Other molecular methods.