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Purification of Antibodies Using Ion-Exchange Chromatography

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Ion-exchange chromatography is a rapid and inexpensive procedure employed to purify antibodies partially from different sources and species (1 4 ). It is a particularly useful tool for isolating antibodies that either do not bind or that bind only weakly to protein A (e.g., mouse IgG1 ) (3 ). This purification method should not be used alone to obtain purified immunoglobulins from crude starting material, but should either be preceded by ammonium sulfate fractionation (see Chapter 2 ) or followed by affinity chromatography (see Chapter 4 ). The principles and theory of ion-exchange chromatography are discussed in detail by Himmelhoch (5 ), and in reference (6 ). Ion-exchange chromatography separates proteins according to their surface charge. Therefore, this separation is dependent on the pI of the protein of interest, the pH and salt concentration of the buffer, and on the charge of the stationary ion-exchange matrix. Proteins are reversibly bound to a charged matrix of beaded cellulose, agarose, dextran, or polystyrene. This interaction can be disrupted by eluting with increasing ionic strength or a change in pH. An ion-exchange matrix should be stable, have good flow characteristic, and not interact nonspecifically with proteins.
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