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Affinity purification

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

Batch purification

1. The affinity matrix is added to a tube and equilibrated with an appropriate buffer.

2. Then, the protein sample is added. The tube is gently rotated or shaken during incubation to prevent that the beads settle.

3. Next, the beads are separated e.g. via centrifugation, and the supernatant is removed.

Note: Multiple washing steps are usually required before elution of the target protein.

Batch purification is primarily used for small-scale purifications, for purifying multiple test expressions at the same time (e.g. 96 well format) or for purification of proteins from large volumes (e.g. purification of low expressed secreted proteins from cell culture supernatants).


Affinity chromatography

For affinity chromatography, the matrix is packed either into gravity flow column, or alternatively, an FPLC (Fast Protein Liquid Chromatography) column.

For gravity flow affinity chromatography, the affinity resin is filled into a column. After the beads have settled, the resin is equilibrated with an appropriate buffer. Next, cellular lysate is applied to the column. Most proteins run through the column, while the protein of interest is retained. After washing the column with several column volumes of washing buffer, the protein of interest can be eluted and collected.


Nanobodies for affinity purification

Capture ligands with high specificity and stability are needed for affinity purification. Nanobodies are especially well suited for affinity purification, as they combine both high selectivity with a high degree of robustness. Since Nanobodies are very stable and remain functional even under extreme buffer conditions, highly stringent washing protocols (e.g. detergent, low pH, high salt etc.) and multiple regeneration cycles of the affinity resin can be applied. Accordingly, they are perfect tools for affinity purification.


Affinity purification of Spot-tagged proteins

Spot-tagged proteins are purified using Spot-Cap® (i.e. anti-Spot Nanobody covalently coupled to beads). Bound proteins can be eluted at acidic pH, or, more gently, by competitive elution using free Spot peptide. The high stability of the anti-Spot Nanobody is reflected by an extraordinarily robust performance of the Spot-Trap in the presence of detergents, chaotropic agents, high salt concentrations or extreme pH. Even multiple regeneration cycles for repeated use of the Spot-Cap are possible.

The Spot-Tag is an inert 12 amino acid peptide-tag (PDRVRAVSHWSS), which is specifically bound by the highly stable and robust anti-Spot-Nanobody used in Spot-Cap.



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