Introduction

The term affinity chromatography began to be used extensively in the 1960s to describe protein separation methods that made use of the speciRc biological interaction of the desired protein with some ligand that was immobilized on an adsorbent matrix. Since most proteins, and all enzymes, bind to some compound very speciRcally, this immediately promised to solve most protein puriRcation problems. But, as with all good ideas, there were many cases when it did not work as expected; the general concept of affinity chromatography for purifying proteins found its niche, but was no panacea. More recently, it has found a fairly widespread application in purifying recombinant proteins, using very standardized procedures.

Although most applications have been for proteins, it is not so limited in theory, since other biological macromolecules have speciRc interactions which can be exploited, especially nucleic acids. But, for the purposes of this article, the principles will be expounded with proteins as the prime target. We should consider the deRnition(s) of afRnity chromatogra-phy carefully, since it does not mean the same to everyone. First, the word afRnity. Any two components that are attracted to each other can be said to have an afRnity, but if we took that as a deRni-tion, the term would be too broad to be useful - for instance, it could include all types of chromatogra-phy. It is better to limit the definition of affinity to a biologically significant interaction such as between a hormone and its receptor, an enzyme and its substrate, or an antibody and its antigen. Unfortunately, there are well-established uses of the term, such as immobilized metal affinity chromatography, in which

Starkenstein EV (1910) Über fermentenwir-kungund deren beeinflussung durch neutralsalze. Biochemische Zeitschrift 24: 14.

Teng SF, Sproule K, Hussain A and Lowe CR (1999) A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial; synthesis and biological evaluation of an IgG binding ligand. Journal of Molecular Recognition 12: 67.

Tuerk C and Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249: 505.

the interaction is not biologically relevant, though it too can be highly speciRc. Perhaps a better deRnition could imply simply a high speciRcity and selectivity of the interaction, though that can exclude some examples of true biological afRnity.

The other word, chromatography, strictly means that process in which components are adsorbed and desorbed continuously as they move down a column, or through some other medium, resulting in a multistage separation of different components according to their partitioning between the stationary and mobile phases. But afRnity methods are usually treated in an 'on-off' fashion in which, after total adsorption of the desired component, a stepwise change in the buffer mobile phase results in its complete elution, and true chromatography is not carried out. Nevertheless, the word chromatography is used more widely than its strict deRnition, to encompass any use of an adsorbent, even in this 'on-off' fashion.

And so we come up with a deRnition of afRnity chromatrography as a chromatographic procedure utilizing an adsorbent involving an immobilized ligand which has a high speciRcity for binding the desired component, preferably to the exclusion of all others. This binding can be loosened by a change in buffer conditions, to elute the desired component relatively free of contaminants. If the ligand is the natural biological ligand of the desired component, then the more precise term bioafRnity chrom-atography can be used. On the other hand, when the interaction is speciRc, but the ligand is unnatural, terms such as pseudo-afRnity chromatography and biomimetic chromatography have been adopted.

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Solar Panel Basics

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