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Date: 19-4-2016
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Affinity chromatography
The chromatographic methods discussed above are all dependent upon the gross physicochemical properties of the protein. However, the biological activity of the protein is generally more subtle and depends upon the very specific, complementary, steric relationship between the active site and a substrate (or inhibitor), or a binding site and a ligand, as the case may be. Affinity chromatography exploits this biospecific relationship between a protein and a ligand, to specifically select out a desired protein from a crude mixture, essentially in a single step.
The specific ligand, which in the case of an enzyme may be a substrate or an inhibitor, is immobilized by conjugation to an insoluble matrix, in a manner which does not interfere with its interaction with the protein.
This may require the use of a spacer arm, which typically consists of a chain of about 6-10 carbon atoms. An affinity chromatography resin is thus comprised of three parts, i) the matrix, which is similar to the matrices used for ion-exchange chromatography, ii) a spacer arm, and iii) the ligand. Matrix/spacer arm combinations are commercially available, since these are universal reagents, and simply require the addition of an appropriate ligand.
The sample solution is passed through the column and by its specific interaction with the immobilized ligand the protein of interest is retained, while all other proteins pass straight through the column. Subsequently, the protein can be eluted by a change in either the pH or ionic strength of the buffer or by addition of a free competing ligand, or of a chaotrope. Because the protein is immobilized in a small volume of resin, affinity columns are generally quite small. Also, the volume of solution in which the protein occurs may be large and to pass this volume through the
References
-Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .
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