Immunoaffinity Column Application

1. Pack the gel into a plastic or other suitable column after allowing the gel to warm to room temperature and being careful to keep the column free of air bubbles or channels. Normally, a visual assessment for air bubbles or channels is adequate; if air bubbles are present then the column may be gently tapped against a hard surface or the column may be repacked. We routinely pack 1 mL of immunoaffinity gel into 5-mL columns equipped with a bottom frit (200-300 ^m) to retain the gel. We do not normally use a top frit although other researchers find this protects the column.

2. Carefully apply the sample (see Notes 4 and 5) to the column. The sample may be applied using gravity flow or with a peristaltic pump, but use care not to disrupt the gel in the column. Theoretically, there is no limit to the volume of sample added, but the sample amount should not exceed the binding capacity of the column.

3. Wash the column with a solution that is strong enough to wash off the interferences but not the analyte(s). We commonly use 10-col-umn volumes of 10% MeOH in water. Column washing is a crucial step in the process because it is the step in which most interference are removed while the analyte remains bound by the immobilized antibody. Determination of an appropriate wash solution is an empirical process.

4. Elute the analyte (see Note 6) by applying 10-column volumes of 50 mM glycine HCl, pH 2.8, or another eluent sufficiently strong to remove the analyte, but mild enough to avoid damage to the column. Again this is a critical step, because you wish to elute the analyte and nothing else. In addition, the integrity of the immobilized antibody on the column should be preserved to allow reuse of the column. We have found the glycine buffer works well to remove the analyte without damaging the immobilized antibody. Again, the user may need to experiment with other solutions dependent on the particular antigen and antibody they are using. The analyte-antibody interaction is controlled by nonbonded interactions, that is, hydrogen bonds, van der Waals forces, ionic interactions, and hydrophobic bonding. The solution that elutes the analyte must disrupt these forces, so changes in pH, ionic strength, or increased organic solvent content have been used.

5. Regenerate the column by applying five-column volumes of 0.1 M Tris-HCl pH 8.0/0.5 M NaCl followed by five-column volumes of 0.1 M acetate buffer pH 4.0/0.5 M NaCl. This cycle of a two buffer wash is repeated an additional two times. It is important to remove all analyte from the column to prevent carryover during column reuse.

6. Store the column at 4°C in PBS, pH 7.2, with 0.02% sodium azide.

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