Affinity Chromatography - Department of Molecular and Cellular ...
Affinity Chromatography - Department of Molecular and Cellular ...
Affinity Chromatography - Department of Molecular and Cellular ...
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M r<br />
97 000<br />
66 000<br />
45 000<br />
30 000<br />
20 100<br />
14 000<br />
Lane 1.<br />
Lane 2.<br />
Lane 3.<br />
Lane 4.<br />
Lane 5.<br />
Lane 6.<br />
Lane 7.<br />
Low <strong>Molecular</strong> Weight Calibration Kit<br />
Egg yolk extract<br />
Flow-through pool<br />
Eluted IgY<br />
Egg yolk extract, diluted 4-fold<br />
Flow-through pool, diluted 4-fold<br />
Eluted IgY, diluted 4-fold<br />
1 2 3 4 5 6 7<br />
Fig. 21. SDS-PAGE <strong>of</strong> non-reduced samples on PhastSystem, using PhastGel 4–15%, Coomassie staining.<br />
Performing a separation<br />
Column:<br />
Recommended flow rate:<br />
HiTrap IgY Purification HP<br />
5 ml/min<br />
Binding buffer: 20 mM sodium phosphate, 0.5 M K 2 SO 4 , pH 7.5<br />
Elution buffer: 20 mM sodium phosphate, pH 7.5<br />
Wash buffer:<br />
20 mM sodium phosphate, pH 7.5 with 30% isopropanol<br />
As much as possible <strong>of</strong> the egg yolk lipid must be removed before purification. Water or<br />
polyethylene glycol can be used to precipitate the lipids. Precipitation with water is<br />
described below.<br />
Precipitation <strong>of</strong> the egg yolk lipid using water<br />
1. Separate the egg yolk from the egg white.<br />
2. Add nine parts <strong>of</strong> distilled water to one part egg yolk.<br />
3. Mix <strong>and</strong> stir slowly for 6 hours at +4 °C.<br />
4. Centrifuge at 10 000 g, at +4 °C for 25 minutes to precipitate the lipids.<br />
5. Collect the supernatant containing the IgY.<br />
6. Slowly add K 2 SO 4 to the sample, stirring constantly, to reach a concentration <strong>of</strong> 0.5 M.<br />
7. Adjust pH to 7.5.<br />
8. Pass the sample through a 0.45 µm filter immediately before applying it to the column.<br />
Purification<br />
1. Wash the column with at least 5 column volumes <strong>of</strong> binding, elution <strong>and</strong> wash buffer.<br />
2. Equilibrate with 5 column volumes <strong>of</strong> binding buffer.<br />
3. Apply the sample.<br />
4. Wash with at least 10 column volumes <strong>of</strong> binding buffer or until no material appears in the eluent,<br />
as monitored at A 280 .<br />
5. Elute the IgY with 10 column volumes <strong>of</strong> elution buffer.<br />
6. Wash the column with 8 column volumes <strong>of</strong> wash buffer.<br />
7. Immediately re-equilibrate the column with 5 column volumes <strong>of</strong> binding buffer.<br />
41