Immunoprecipitation and  
Affinity Chromatography


Immunoprecipitation is an application of antibodies that allows identification and characterization of antigens as well as third party proteins that associate with the antigen under physiological conditions. In the early days of antibody biology, immunoprecipitation referred to incubation of polyclonal antiserum with relatively large antigens, that allowed a lattice-like "immunoprecipitate" to form through crosslinking between antigen and antibody. This immunoprecipitate could be separated from the sample by centrifugation and characterized. Nowadays, all types of antibodies can be used to isolate antigens and antigen-binding proteins by conjugation of the antibody to macromolecular beads or absorption of the antibody onto beads that have been derivatized with antibody Fc-binding proteins such as protein A or protein G. These beads can easily be separated from the rest of the sample by centrifugation, filtration, or, if ferromagnetic, by a magnet or magnetic field. Immunoprecipates are typically analyzed by gel fractionation methodologies, followed by simple staining procedures, autoradiography, or Western Blotting.

In a related application known as Affinity Chromatography, macromolecular resins derivatized with antibody or antibody Fc-binding proteins are used to bind antigen from applicable biological samples such as serum, cell/tissue lysate or conditioned medium. The resin is then packed into an affinity column that is washed with an appropriate buffer, before it is eluted with a solution that dissociates antigen from antibody such as low or high pH, hapten, chaotropic ions, etc. Affinity chromatography is being used extensively in biomedical research as well as in large scale production of recombinant therapeutics.
Antibody Solutions has generated many antibodies for immunoprecipitations and affinity chromatography applications and has developed optimized protocols.

Left we depict an immunoprecipitation of human vasointestinal peptide receptor-1 (VIPR1) from metabolically radiolabeled CHO cells transfected with plasmids encoding epitope-tagged VIPR1 (V5 at N-terminus and Xpress at C-terminus). In this experiment, our monoclonal VIPR1-specific antibody AS58-P was compared to two commercial epitope-tag specific antibodies, anti-V5 and anti-Xpress. The amount and purity of recombinant VIPR1 precipitated by our VIPR1-specific antibody at the appropriate molecular weight was equal if not superior to that precipitated by the epitope tag-specific control antibodies and indicates that AS58-P can immunoprecipitate VIPR1 from cells.

Antibody Solutions offers a wealth of expertise in developing antibodies for immunoprecipitations and affinity chromatography applications. Please contact us with your specific application needs.

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