Affinity Chromatography Handbook

32
Performing a separation
Column: HiTrap Protein G HP, 1 ml
Recommended flow rate: 1 ml/min
Binding buffer: Dilute buffer concentrate 10-fold
Elution buffer: Dilute buffer concentrate 10-fold
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 g for 10 minutes) to remove cells and debris. Filter through a
0.45 µm filter. If required, adjust sample conditions to the pH and ionic strength of the
binding buffer either by buffer exchange on a desalting column (see page 133) or by
dilution and pH adjustment.
A B C
Fig. 16. Using HiTrap Protein G HP with a syringe. A: Dilute buffers and prepare sample. Remove the column’s top cap
and twist off the end. B: Equilibrate the column, load the sample and begin collecting fractions. C: Wash and elute,
continuing to collect fractions.
1. Allow the column and buffers to warm to room temperature.
2. Dilute the binding and elution buffers.
3. Connect the syringe to the column using the luer adapter supplied.
4. Equilibrate the column with 5 ml distilled water, followed by 3 ml diluted binding buffer.
5. Apply the sample.
6. Wash with 5–10 ml diluted binding buffer until no material appears in the eluent.
7. Elute with 3–5 ml diluted elution buffer. Collect fractions into tubes containing neutralization buffer.
8. Immediately re-equilibrate the column with 5 ml diluted binding buffer.