This web page was produced as an assignment for an undergraduate course at Davidson College.

Affinity Chromatography

in progress


The idea for purifying enzymes using other substrates and/or bioligands specifically chosen for their bioselective affinity is not a new concept. As early as 1919, scientists were noticing that certain substances bound very strongly to certain enzymes (Scouten, 1981). Along with this, lectins, recognized to bind to sugars and certain cells, have been used in blood typing for the greater part of the 1900's. However, it was not until 35 years ago, that truly marked the beginning of what is today considered affinity chromatography. Since Cuatrecasas's experiment (Cuatrecasas,1968), there has been a tremendous growth in the field of affinity chromatography.


What is Affinity Chromatography (AC):
Affinity chromatography is the purification of a biomolecule with respect to the specific binding of that biomolecule due to its chemical structure (Amersham Bioscience, 2003). With this structure, the biomolecule can reversibly bind to a ligand (Figure 1) which has formed covalent bonds with a chromatographic bed material (matrix) (Amersham Bioscience, 2003).

© Amersham Biosciences Limited - All rights reserved

Fig 1. Binding receptors of a ligand demonstrating the reverse binding ability of its receptors. This property enables the ligand to bind to a biomolecule until washing, at which time the unbound biomolecules are washed away.

Since only the intended biomolecule has bound to the ligands, the unbound substances can then be washed away (Amersham Bioscience, 2003). After the unbound substances are washed away, the bound biomolecule can then be eluted by changing the experimental conditions so that the biomolecule will release from the ligand (Amersham Bioscience, 2003). Another way to elute the bound biomolecule is through the addition of pressure (Parikh, 1993). It has been known that the addition of pressures between 1000 and 5000 atm to proteins in solution will cause them to break apart the individual subunits of a larger protein, without denaturation (Parikh, 1993). However, pressures of 6000 atm or more may cause irreversible denaturation (Parikh, 1993).

-Flash movie of affinity chromatography(provided by Amersham Bioscience)

-More on how AC purifies



In recent years some of the most exciting applications for affinity chromatography have arisen, particularly in the area of recombinant DNA technology. Not until recently, however, has the technology been defined to allow isolation of DNA-binding proteins (Parikh, 1993). While still looking at DNA technology, some nuclear proteins such as histones, non-histones and chromosomal proteins are gaining a large degree of that attention. Isolation and characterization of some nonhistone proteins has gained particular attention due to these cells having control over many transcription processes (Parikh, 1993). Still, the main area of concentration for affinity chromatography applications continues to be in protein purification (Parikh, 1993).



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