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Technique of precipitating a protein antigen out of solution using an antibody that specifically binds to the particular protein, so as to isolate and concentrate a particular protein from a sample containing different types of proteins.

Traditional immunoprecipitation (IP) method

Crosslink immunoprecipitation (IP) method

Involves incubation of antibody with a protein mixture containing the antigen, followed by capture of the antibody - antigen complex to Protein A or Protein G immobilized on beaded agarose resin. After washing to remove nonbound (presumably undesired) components of the sample, antibody and antigen are recovered by boiling the beaded affinity resin in sample loading buffer for analysis by SDS-PAGE.

Uses the principle of crosslinking to covalently attach the antibody to the Protein A/G Agarose Resin – covalent immobilization of the antibody to the agarose resin, enabling purification of antigens without the usual co-elution of the antibody heavy and light chain fragments.

Using the traditional immunoprecipitation (IP) method, both the target antigen and the antibody are eluted in the final fraction, resulting in the presence of prominent heavy and light chain antibody bands in the final electrophoresis gel. If the target protein has a similar molecular weight to one of the contaminating antibody fragments, its presence may be masked, making it impossible to identify or further characterize the immunoprecipitated product.However using the crosslink immunoprecipitation (IP) method, only antigen is eluted by the procedure, enabling it to be identified easily (only one protein band produced) and further analyzed without interference from antibody fragments i.e. eliminating antibody contamination as antibody is irreversibly attached to agarose beads so that co-elution of heavy and light chains with purified protein (antigen) is minimized. It also increase yield of the method and antibodies used may be reused again.

Example.

However, there are certain atypical (uncommon) circumstances that the traditional immunoprecipitation (IP) method works better than the crosslink immunoprecipitation (IP) method:

Antibody used is very unstable and can be inactivated by the covalent crosslinking step. Therefore as a result, there will little/no antigen precipitated despite committing ample (sufficient/excess) antibody to the procedure.

The antibody:antigen affinity interaction are too strong that elution buffer does not efficiently dissociate the target protein (antigen) from the covalently immobilized antibody – harsher elution conditions cannot be used, which may have undesirable denaturing effects to the protein (antigen) of interest.

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