Lectin affinity chromatography

Affinity chromatography is a method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.It is a type of chromatographic lab technique used for purifying biological molecules within a mixture by exploiting molecular properties. Biological macromolecules such as enzymes and other proteins, interact with other molecules with high specificity through several different types of bonds and interaction. Such interactions including hydrogen bonding, ionic interaction, disulfide bridges, hydrophobic interaction, and more. The high selectivity of affinity chromatography is caused by allowing the desired molecule to interact with the stationary phase and become trapped within the column in order to be separated from the undesired material which will not interact and elute first.

Affinity chromatography can be used to:

The stationary phase is typically a gel matrix, often of agarose; a linear sugar molecule derived from algae. Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate, growth medium or blood serum. The molecule of interest will have a well known and defined property, and can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in the mobile phase will not become trapped as they do not possess this property. The stationary phase can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution. Possibly the most common use of affinity chromatography is for the purification of recombinant proteins.

Binding to the solid phase may be achieved by column chromatography whereby the solid medium is packed onto a column, the initial mixture run through the column to allow setting, a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. These steps are usually done at ambient pressure. Alternatively, binding may be achieved using a batch treatment, for example, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase, removing the liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the elute.

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