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for the isolation any GST-tagged cloned protein. It is possible to remove the GST tag

in a one-step process by adding PreScission™Protease to the matrix, as it will be

bound to the column and remove the tag as the protein is eluted.

Practical procedure

The procedure for affinity chromatography is similar to that used in other forms of

liquid chromatography. The buffer used must contain any cofactors, such as metal

ions, necessary for ligand–macromolecule interaction. Once the sample has been applied

and the macromolecule bound, the column is eluted with more buffer to remove non-

specifically bound contaminants. The purified compound is recovered from the ligand

by eitherspecificornon-specific elution. Non-specific elution may be achieved by a

change in either pH or ionic strength. pH shift elution using dilute acetic acid or

ammonium hydroxide results from a change in the state of ionisation of groups in the

ligand and/or the macromolecule that are critical to ligand–macromolecule binding.

A change in ionic strength, not necessarily with a concomitant change in pH, also

causes elution due to a disruption of the ligand–macromolecule interaction; 1M NaCl

is frequently used for this purpose.

Table 11.5Examples of group-specific ligands commonly used in affinity
chromatography

Ligand Affinity

Nucleotides

50 -AMP NADþ-dependent dehydrogenases, some kinases

2050 -ADP NADPþ-dependent dehydrogenases

Calmodulin Calmodulin-binding enzymes

Avidin Biotin-containing enzymes

Fatty acids Fatty-acid-binding proteins

Heparin Lipoproteins, lipases, coagulation factors, DNA polymerases, steroid receptor
proteins, growth factors, serine protease inhibitors

Proteins A and G Immunoglobulins

Concanavalin A Glycoproteins containinga-D-mannopyranosyl anda-D-glucopyranosyl residues

Soybean lectin Glycoproteins containingN-acetyl-a-(orb)-D-galactopyranosyl residues

Phenylboronate Glycoproteins

Poly(A) RNA containing poly(U) sequences, some RNA-specific proteins

Lysine rRNA

Cibacron Blue
F3G-A

Nucleotide-requiring enzymes, coagulation factors

468 Chromatographic techniques