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Affinity Chromatography - Department of Molecular and Cellular ...

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Performing a separation<br />

Column:<br />

HiTrap Protein G HP, 1 ml or 5 ml<br />

Recommended flow rates: 1 ml/min (1 ml column) or 5 ml/min (5 ml column)<br />

Binding buffer: 0.02 M sodium phosphate, pH 7.0<br />

Elution buffer: 0.1 M glycine-HCl, pH 2.7<br />

Neutralization buffer: 1 M Tris-HCl, pH 9.0<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 or by dilution <strong>and</strong> pH adjustment (see<br />

page 134).<br />

1. Equilibrate column with 5 column volumes <strong>of</strong> binding buffer.<br />

2. Apply sample.<br />

3. Wash with 5–10 column volumes <strong>of</strong> the binding buffer to remove impurities <strong>and</strong> unbound material. Continue<br />

until no protein is detected in the eluent (determined by UV absorbance at 280 nm).<br />

4. Elute with 5 column volumes <strong>of</strong> elution buffer*.<br />

5. Immediately re-equilibrate with 5–10 column volumes <strong>of</strong> binding buffer.<br />

*Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (60 µl – 200 µl<br />

1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH <strong>of</strong> the fractions will be approximately neutral.<br />

IgGs from most species <strong>and</strong> subclasses bind to protein G at near physiological pH <strong>and</strong> ionic<br />

strength. For the optimum binding conditions for IgG from a particular species, it is worth<br />

consulting the most recent literature. Avoid excessive washing if the interaction between the<br />

protein <strong>and</strong> the lig<strong>and</strong> is weak, since this may decrease the yield.<br />

Most immunoglobulin species do not elute from Protein G Sepharose until pH 2.7 or less.<br />

If biological activity <strong>of</strong> the antibody or antibody fragment is lost due to the low pH<br />

required for elution, try Protein A Sepharose: the elution pH may be less harsh.<br />

Desalt <strong>and</strong>/or transfer purified IgG fractions to a suitable buffer using a desalting column<br />

(see page 134).<br />

Reuse <strong>of</strong> Protein G Sepharose depends on the nature <strong>of</strong> the sample <strong>and</strong> should only be<br />

performed with identical samples to prevent cross-contamination.<br />

To increase capacity, connect several HiTrap Protein G HP columns (1 ml or 5 ml) in series.<br />

HiTrap columns can be used with a syringe, a peristaltic pump or connected to a liquid<br />

chromatography system, such as ÄKTAprime. For greater capacity pack a larger column<br />

with Protein G Sepharose 4 Fast Flow (see Appendix 3).<br />

30

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