Encyclopedia of Experiments: Immunology
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Transcript
Take an FPLC column containing a porous glass resin with immobilized Protein A — an antibody-binding bacterial protein. Equilibrate the column with a neutral pH buffer.
Add cell culture fluid harvested from Chinese hamster ovarian cells, containing monoclonal antibodies and contaminating proteins and DNA.
Protein A binds to the antibody's Fc region, immobilizing them.
Some contaminants non-specifically attach to the resin, while the rest flow through.
In the chromatogram, an increased UV absorbance during sample loading indicates a high protein concentration in the flow through.
Wash with the equilibration buffer to remove unbound contaminants, indicated by a decrease in the absorbance.
Flush with a high-salt buffer, disrupting the non-specific interactions and removing the contaminants, indicated by a small impurity peak.
Add a low-pH elution buffer to disrupt the Protein A-antibody interaction and elute the antibodies, which are collected by tracking the corresponding peak.
Neutralize the pH of the collected antibodies to prevent degradation.
