Protein Protocols Protein Protocols

Short Chapter Title 1003
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Purification of IgG Using Thiophilic Chromatography
Mark Page and Robin Thorpe
1. Introduction
Immunoglobulins recognize sulphone groups in close proximity to a thioether group
(1), and, therefore, thiophilic adsorbents provide an additional chromatographic method
for the purification of immunoglobulins that can be carried out under mild conditions
preserving biological activity. A thiophilic gel is prepared by reducing divinylsulfone
(coupled to Sepharose 4B) with β-mercaptoethanol. The product is of intermediate
purity and would be useful for further processing, e.g., purification by ion-exchange,
size exclusion, and/or affinity chromatography.
2. Materials
1. Sepharose 4B.
2. 0.5 M sodium carbonate.
3. Divinylsulfone.
4. Coupling buffer: 0.1 M sodium carbonate buffer, pH 9.0.
5. β-mercaptoethanol.
6. Binding buffer: 0.1 M Tris-HCl, pH 7.6, containing 0.5 M K2SO4. 7. IgG preparation: serum, ascitic fluid, or hybridoma culture supernatant.
8. 0.1 M ammonium bicarbonate.
3. Methods
Caution: Divinylsulfone is highly toxic and the column preparation procedures
should be carried out in a well-ventilated fume cabinet.
1. Wash 100 mL of Sepharose 4B (settled volume) with 1 L of water by vacuum filtration.
2. Resuspend in 100 mL of 0.5 M sodium carbonate, and stir slowly.
3. Add 10 mL of divinylsulfone dropwise over a period of 15 min with constant stirring.
After addition is complete, slowly stir the gel suspension for 1 h at room temperature.
4. Wash the activated gel thoroughly with water until the filtrate is no longer acidic (see Note 1).
5. Wash activated gel with 200 mL of coupling buffer using vacuum filtration and resuspend
in 75 mL of coupling buffer.
6. In a well-ventilated fume cabinet, add 10 mL of β-mercaptoethanol to the gel suspension
with constant stirring, and continue for 24 h at room temperature (see Note 2).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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