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New PDF release: Antibody Purification

By Amersham Biosciences

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For routine purification it may be worth developing an immunospecific purification with an anti-IgA monoclonal antibody coupled to a pre-activated affinity matrix to provide a high resolution, high selectivity affinity purification medium (see page 45). Alternatively, a multi-step purification strategy could be employed (see Chapter 5). IgD Protein A and protein G do not bind to IgD. For routine purification it may be worth developing an immunospecific purification with an anti-IgD monoclonal antibody coupled to a pre-activated affinity matrix to provide a high resolution, high selectivity affinity purification medium (see page 45).

Neutralization buffer: Add 60–200 µl of neutralization buffer per ml fraction to the test tubes in which IgG will be collected. Centrifuge samples (10 000 x g for 10 minutes) to remove cells and debris. 45 µm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer by either buffer exchange on a desalting column (see page 21) or by dilution and pH adjustment. 34 1. Allow the HiTrap Protein G HP column and buffers to warm to room temperature. 2. Dilute the buffers.

Remove the twist-off end. Ligand coupling 1. Wash out the isopropanol with 3 x 2 column volumes of ice-cold 1 mM HCl. 2. Inject one column volume of ligand solution onto the column. 3. Seal the column and leave for 15–30 minutes at +25 °C (or 4 hours at +4 °C). Do not use excessive flow rates (maximum recommended flow rates are 1 ml/min, equivalent to approximately 30 drops/min when using a syringe, with HiTrap 1 ml and 5 ml/min, equivalent to approximately 120 drops/min when using a syringe, with HiTrap 5 ml).

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