Protein Protocols Protein Protocols

IgG Purification with Ion-Exchange HPLC 989
140
Purification of IgG Using Ion-Exchange HPLC
Carl Dolman, Mark Page, and Robin Thorpe
1. Introduction
Conventional ion-exchange chromatography separates molecules by adsorbing proteins
onto the ion-exchange resins that are then selectively eluted by slowly increasing
the ionic strength (this disrupts ionic interactions between the protein and column
matrix competitively) or by altering the pH (the reactive groups on the proteins lose
their charge). Anion-exchange groups (such as diethylaminoethyl; [DEAE]) covalently
linked to a support matrix (such as Sepharose) can be used to purify IgG in which the
pH of the mobile-phase buffer is raised above the pI or IgG, thus allowing most of the
antibodies to bind to the DEAE matrix. Compare this method with that described in
Chapter 139 in which the IgG passes through the column. The procedure can be carried
out using a laboratory-prepared column that is washed and eluted under gravity (1);
however, high-performance liquid chromatography (HPLC) provides improved reproducibility
(because the sophisticated pumps and accurate timers), speed (because of the
small high-capacity columns), and increased resolution (because of the fine resins and
control systems).
2. Materials
1. Anion-exchanger (e.g., Mono-Q HR 5/5 or HR 10/10, Pharmacia, Uppsala, Sweden.
2. Buffer A: 0.02 M triethanolamine, pH 7.7.
3. Buffer B: Buffer A containing 1 M NaCl.
4. 2 M NaOH.
5. Sodium azide.
3. Methods
1. Prepare serum by ammonium sulfate precipitation (45% saturation; see Chapter 137).
Redissolve the precipitate in 0.02 M triethanolamine buffer, pH 7.7, and dialyze overnight
against this buffer at 4°C. Filter the sample (see Note 1) before use (0.2 µm).
2. Assemble the HPLC system according to the manufacturer’s instructions for use with the
Mono-Q ion-exchange column.
3. Equilibrate the column with 0.2 M triethanolamine buffer, pH 7.7 (buffer A). Run a blank
gradient from 0 to 100% buffer B (buffer A + 1 M NaCl). Use a flow rate of 4–6 mL/min
for this and subsequent steps.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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