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Affinity chromatography


Affinity chromatography is a biochemical separation method that combines size fractionation capability of gel permeation chromatography with the ability to design a stationary phase that reversibly binds to a known subset of molecules. This method is usually used to:

  • Purify and concentrate a molecule from a mixture into a buffering solution.
  • Reduce the amount of a molecule in a mixture.
Contents

General Procedure

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 which can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule (ie the molecule targeted for purification) becoming trapped on a solid or stationary phase or medium. The non-target heterogeneous mixture will not become trapped. The solid medium can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution. The process can be illustrated thus:


Of course if the object is to remove an undesirable molecule from the initial mixture, then once the initial binding has been achieved, the material of interest is the unbound mixture, and the washing and elution steps can be omitted or used simply for analytical purposes.

Specific Uses

Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts and antibody purification from blood serum.

Possibly the most common use of affinity chromatography is for the purification of recombinant proteins. Essentially proteins are tagged with a known affinity ligand in order to aid their purification. That is to say that the molecule has an artificial trait imposed upon it by genetic modification which allows it to be selected for affinity binding (if the molecule is a protein these are called fusion proteins). Such tags include six His tags (six Histidine residues added to one or both ends of a protein) and GST (glutathione-S-transferase) tags. Six His tags have an affinity for nickel ions which are covalently bound to NTA for the purposes of solid medium entrapment [[1]]. For elution purposes a nickel chelating agent is used. GST has an affinity for glutathione (commercially available as glutathione sepharose ). For elution purposes glutathione is used.

Another use for the procedure is the affinity purification of antibodies from blood serum. If serum is known to contain antibodies against a specific antigen (for example if the serum comes from an organism immunised against the antigen concerned) then it can be used for the affinity purification of the antibody. For example if an organism is immunised against a GST-fusion protein it will produce antibodies against the fusion-protein, and possibly antibodies against the GST tag as well. The protein can then be covalently coupled to a solid medium such as sepharose or agarose (these are commercially available). For thoroughness the GST protein and the GST-fusion protein can each be coupled separately. The serum is initially allowed to bind to the GST affinity matrix, this will cause any anti-GST antibodies to bind (that is any antibodies against the GST part of the fusion protein will be removed). The serum is then separated from the solid medium and allowed to bind to the GST-fusion protein matrix. This allows the remaining antibodies to bind to the GST-fusion protein. Elution is achieved by the use of a low pH buffer such as glycine pH 2.8. The eluate is collected into tris buffer pH 8 as low pH will degrade the antibody. This is a nice example as affinity purification is used to purify the initial GST-fusion protein, to remove the undesirable anti-GST antibodies from the serum and to purify the target antibody.

Batch verses column

Binding to the solid phase may be achieved by column chromatography, whereby the solid medium is packed onto a chromatography column , the initial mixture run through the column to allow binding, 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 (as opposed to HPLC or FPLC )


Alternatively binding may be achieved using a batch treatment, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase (by centrifugation for example), removing the liquid phase, washing, re-centrifuging , adding the elution buffer , re-centrifuging and removing the eluate.


Sometimes a hybrid method is employed, the binding is done by the batch method, then the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column.

Affinity Chromatography Protocols

[Protocol-Online]


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