Question about antibody affinity chromatography

[Sunwoo Bae]

TL;DR Summary

A question about how changing the pH of the buffer allows you to elute the protein that in bonded to the antibody.

I am learning about protein purification in my Biochemistry class, and I have a question about protein elution in antibody affinity chromatography.

My understanding of the mechanism for the technique is that proteins that do not bind to the antibody will be separated first as it runs down the column. The proteins that do bind to the column will be stuck on the antibody, so there should be a way to elute those proteins for protein purification.

I have been taught that in order to separate the proteins bound to the antibody, you need to use a low-pH buffer to detach the proteins from the antibody. Can anyone explain how this works?

Thank you.

 

[chemisttree]

pH can change the tertiary structure of proteins.

 

[jim mcnamara]

Lowering pH has problems and may lower recovery rates due to aggregation. A discussion and alternate method:"
Elution of antibodies from a Protein-A column by aqueous arginine solutions

Tsutomu Arakawa, John S Philo, Kouhei Tsumoto, Ryosuke Yumioka, Daisuke Ejima

Affiliations

• PMID: 15249046
• DOI: 10.1016/j.pep.2004.04.009

Abstract
Acidic pH is commonly used to elute antibodies from Protein-A affinity column, although low pH may result in aggregation of the proteins. As an alternative, here arginine was tested as an eluent and compared with a more conventional eluent of citrate. Using purified monoclonal antibodies, recovery of antibodies with 0.1M citrate, pH 3.8, was less than 50% and decreased further as the pH was increased to 4.3. At the same pH, the recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 31% recovery, although the elution under such condition showed extensive tailing. Such tailing was observed at pH 3.8 when 0.1M citrate was used. Size exclusion analysis indicated that the eluted antibodies were mostly monomeric whether eluted with citrate or arginine. This demonstrates the usefulness of arginine as an efficient eluent for Protein-A chromatograp is probably aware of better methods.

Other members may know more. I posted this to show that there may be other suitable methods, but your lab instructor may want you to learn only about lowering pH.

 

[chemisttree]

From https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-affinity-chromatography.pdfexplaining affinity chromatography...

“A change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein. This change may affect the binding sites directly, reducing their affinity, or cause indirect changes in affinity by alterations in conformation.”