Biochemical/molecular techniques

Background concepts:
Immunoprecipitation

Typically, a blood or urine sample contains a variety of proteins (crude sample) while only one may be of diagnostic interest. Immunoprecipitation is often used to purify a target protein from solution (purified sample). This way, the protein of interest can be further examined for quantity or physical characteristics (SDS-PAGE). This technique involves the interaction between a protein and its specific antibody, the separation of the immune complexes thus formed with a protein G coated resin or support.

• An antibody specific to the target protein is added to the protein mixture. The antigen-antibody complex is then precipitated from the solution using an insoluble resin which binds to the antibody complex. Unbound proteins are removed by washing the resin. The target protein is eluted from the resin for further analysis. However, the antibody is also collected at this point and complicates the analysis of the purified target protein.
• a better method is to attach covalently the antibody to the resin. Then the antibody coupled to resin is added to a crude protein mixture to capture and precipitate the antigen (which will become the purified protein). The antigen is eluted from the resin while the antibody remains covalently bound to the resin. This simplifies the analysis of the purified protein by not having the antibody interfere with further analysis such as SDS-PAGE or protein assays.

The antibody is bound and immobilized to a Protein G support using a cross-linking agent Disuccinimidyl suberate (DSS). This properly orients the antibody to "seize" protein (antigen-antibody complex) from a crude sample applied to the immobilized antibody support.
Protein G is a bacterial cell wall protein isolated from group G streptococci: its use in this technique relies on its unique ability to bind the Fc portion of the antibody and not the Fab fragments of the antibody. Antigens can thus be isolated by incubation on a Protein G column that has had their complementary antibody first bound to the immobilized Protein G.

The antigen is then recovered by elution: a buffer is used to wash and release trapped ions or molecules from the antibody support (protein G coated beads or resin).

The immunoprecipitated protein is then collected for further analysis.