Protein affinity chromatography evaluation

In affinity chromatography, the resin contains especially selected molecules that will interact with the particular polymer(s) that is being studied. Thus, for a particular protein, the resin may be modified to contain a molecule that interacts with that protein type. The solution containing the mixture is passed through the column and the modified resin preferentially associates with the desired protein, allowing it to be preferentially removed from the solution. Later, the protein is washed through the column by addition of a salt solution and collected for further evaluation. 

Hydrophobic chromatography is still a relatively new technique for the biochemist and many fundamental aspects remain to be solved before the true value of these procedures can be evaluated. However, initial experiments indicate that the technique will prove to be another valuable tool in protein separations. In contrast to affinity chromatography, where specific supports must be designed for each application, it appears that a limited number of supports should suffice for a wide range of applications. 

Whole serum can be used in preliminary evaluation of antibody performance, but the final lateral flow assay should be developed with purified antibody. Affinity chromatography with protein A or G can purify total antibodies from the serum, but purification on immobilized antigen is preferred, since it isolates only analyte-specific antibodies (2). Using only antibodies specific to the target helps minimize background and increase assay sensitivity. 

Examine Table 4.1 in the text, evaluating a protein purification scheme. Does total activity go up or down as the protein is purified Would it have been a good idea to try affinity chromatography at an earlier stage of purification ... 

Grisshammer R. and Tucker J. 1997. Quantitative evaluation of neurotensin receptor purification by immobilized metal affinity chromatography. Protein Expr. Purif., 11, 53. 

In conclusion, it may be reasonably assumed that if a protein contains covalentlS linked carbohydrate, then an affinity chromatographic ccdumn based on a lectin or panB of lectins, can be selected for detailed evaluation of the monosaccharide composition sequence and branching patterns. Such lectin affinity chromatography is a rapid, hi resolution non-destructive analytical procedure that can be applied to trace quantiti of material. New lectins are constantly being discovered, so we can safely anticipate the availability of progressively more refined lectin analytical reagents. 

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