Continuation of our series of articles on protein purification techniques : Last February we described IEX- Chromatographie par Échange d’Ions, today we are focusing on affinity.
This technic is the most efficient among all the existing proteins purification technics: it allows to recover proteins pure up to 90%.
The affinity technics port folio is large: Protein A affinity, IMAC affinity (Immobilized Metal Affinity Chromatography), GST-Tag affinity…
Affinity chromatography is the selective separation of active biomolecules, and their isolation from their inactive or denatured forms.
The separation is done by specific interaction between an immobilized ligand & its partner: antibody/antigen, enzyme/substrate, enzyme/inhibitor…
- Perfect selectivity, high purity 80 – 90%
- Purifications in 1 or 2 steps
- Proteins concentration
- Generic purification conditions – Often no need of optimization
- High cost (more particularly protein A)
THE technic of choice for antibodies and recombinant proteins.
Affinity chromatography classical pattern:
How affinity works?
With affinity chromatography, high-selectivity separation of biomolecules can be achieved through their specific interactions. This separation technique is special because it is based on the biological function or the unique chemical structure of a given biomolecule. During affinity chromatography, the interacting partner – the Ligand – of the biomolecule is immobilized on a chromatographic resin. The material is eluted from the column by changing the composition of the mobile phase.
- High selectivity
- High resolution
- High capacity
The degree of purification can be thousands of times, and the achieved yield can also be usually very good.
Affinity chromatography is unique in the sense that it is based on the specific biological function of the biomolecule of interest.
Many separations adsorbants exist for natural or recombinant proteins:
- Protein A for antibodie
- Immobilized Metal Affinity Chromatography (IMAC) for Histidin tag proteins
- Glutathion for GST (Glutathion S Transferase) tag proteins