Protein Protocols Protein Protocols

Antigen-Ligand Columns 999
144
Purification of IgG Using Affinity Chromatography
on Antigen-Ligand Columns
Mark Page and Robin Thorpe
1. Introduction
Affinity chromatography is a particularly powerful procedure, which can be used to
purify IgG, subpopulations of IgG, or the antigen binding fraction of IgG present in
serum/ascitic fluid/hybridoma culture supernatant. This technique requires the production
of a solid matrix to which a ligand having either affinity for the relevant IgG or
vice versa has been bound (1). Examples of ligands useful in this context are:
1. The antigen recognized by the IgG (for isolation of the antigen-specific fraction of the
serum/ascitic fluid, and so forth).
2. IgG prepared from an anti-immunoglobulin serum, e.g., rabbit antihuman IgG serum or
murine antihuman IgG MAb for the purification of human IgG (see Note 1).
3. IgG-binding proteins derived from bacteria, e.g., protein A (from Staphylococcus aureus
Cowan 1 strain) or proteins G or C (from Streptococcus and see Chapter 142).
The methods for production of such immobilized ligands and for carrying out affinity-purification
of IgG are essentially similar, regardless of which ligand is used.
Sepharose 4B is probably the most widely used matrix for affinity chromatography,
but other materials are available. Activation of Sepharose 4B is usually carried out by
reaction with cyanogen bromide (CNBr); this can be carried out in the laboratory before
coupling, or ready-activated lyophilized Sepharose can be purchased. The commercial
product is obviously more convenient than “homemade” activated Sepharose, but it is
more expensive and may be less active.
2. Materials
1. Sepharose 4B.
2. Sodium carbonate buffer: 0.5 M Na2CO3, pH 10.5. Adjust pH with 0.1 M NaOH.
3. Cyanogen bromide. (Warning: CNBr is toxic and should be handled in a fume hood.)
4. Sodium hydroxide: 1 M; 4 M.
5. Sodium citrate buffer: 0.1 M trisodium citrate, pH 6.5. Adjust pH with 0.1 M and citric acid.
6. Ligand solution: 2–10 mg/mL in 0.1 M sodium citrate buffer, pH 6.5.
7. Ethanolamine buffer: 2 M ethanolamine.
8. PBS: 0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4. From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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