Affinity purification is a technology that enriches or purifies a protein from a complex mixture of biomolecules such as a cellular lysate, cell culture supernatant or serum etc. The protein of interest is selectively bound by a ligand like an antibody or Nanobody, which is attached to a matrix, such as agarose beads.

Frequently, the protein of interest is expressed recombinantly and fused to a protein- or peptide-tag, which is bound specifically and reversibly by the affinity resin. Enriched protein can be released either by competitive elution using a molecule like a peptide competing for binding to the affinity resin, or simply by changing the buffer conditions, such as pH shift or change in ionic strength.

Most commonly, affinity purification is done either as batch purification or via a column packed with the affinity resin (affinity chromatography).

Affinity purification comprises 4 steps:

• Equilibration of the affinity resin with an appropriate buffer
• Loading of the sample containing the protein of interest to the affinity resin
• Washing and removal of unwanted proteins and other macromolecules
• Elution of the protein of interest