Technical information > Antibody purification

Antibody purification is advisable for certain applications, such as immunoprecipitation, immunohistochemistry and antibody labeling.

Important notes

• The total immunoglobulin pool will contain only 0.5-10% of antigen-specific antibodies. The remaining antibodies are active against other antigens
• If certain antigen-binding antibodies are lost or an enrichment of non-specific antibodies occurs during the purification process, the signal-to-noise ratio can deteriorate rather than improve

Affinity purification

To obtain antigen-specific antibodies (from serum or IgY fraction), the following steps can be performed:

• Coupling of peptide or other antigen to matrix. UltraLink® can be used for peptides, and Hi trap NHS for proteins. The affinity column can be reused
• Antigen-specific purification
• Specific activity of affinity-purified antibodies confirmed by ELISA

The theoretical yield from affinity purification is around 0.5-5 mg of antigen-specific antibodies per 1 mg of peptide coupled to the support, or 0.5-2 mg of antigen-specific antibodies per 1 mg of protein coupled to the support.

Protein A/G purification

To obtain total immunoglobulin fraction, the following steps can be performed:

• Isolation of total IgG from 20 ml serum (rabbit, goat, mouse and rat) using Protein G Sepharose 4 Fast Flow
• Specific activity of affinity-purified antibodies confirmed by ELISA

In case of very low amounts of protein are available for affinity purification, it can be done using this approach.