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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Performing a separation<br />
Column:<br />
Recommended flow rate:<br />
Binding buffer:<br />
Elution buffer:<br />
Neutralization buffer:<br />
HiTrap Protein G HP, 1 ml<br />
1 ml/min<br />
Dilute buffer concentrate 10-fold<br />
Dilute buffer concentrate 10-fold<br />
Add 60–200 µl <strong>of</strong> neutralization buffer per ml fraction to the test tubes in which IgG<br />
will be collected<br />
Centrifuge samples (10 000 g for 10 minutes) to remove cells <strong>and</strong> debris. Filter through a<br />
0.45 µm filter.<br />
If required, adjust sample conditions to the pH <strong>and</strong> ionic strength <strong>of</strong> the binding buffer<br />
either by buffer exchange on a desalting column (see page 134) or by dilution <strong>and</strong> pH<br />
adjustment.<br />
A B C<br />
Fig. 16. Using HiTrap Protein G HP with a syringe. A: Dilute buffers <strong>and</strong> prepare sample. Remove the column’s top cap<br />
<strong>and</strong> twist <strong>of</strong>f the end. B: Equilibrate the column, load the sample <strong>and</strong> begin collecting fractions. C: Wash <strong>and</strong> elute,<br />
continuing to collect fractions.<br />
1. Allow the column <strong>and</strong> buffers to warm to room temperature.<br />
2. Dilute the binding <strong>and</strong> elution buffers.<br />
3. Connect the syringe to the column using the luer adapter supplied.<br />
4. Equilibrate the column with 5 ml distilled water, followed by 3 ml diluted binding buffer.<br />
5. Apply the sample.<br />
6. Wash with 5–10 ml diluted binding buffer until no material appears in the eluent.<br />
7. Elute with 3–5 ml diluted elution buffer. Collect fractions into tubes containing neutralization buffer.<br />
8. Immediately re-equilibrate the column with 5 ml diluted binding buffer.<br />
32