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Affinity Chromatography - Department of Molecular and Cellular ...

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Gradient <strong>and</strong> step elution<br />

Figure 6 shows examples <strong>of</strong> step <strong>and</strong> gradient elution conditions. For prepacked affinity<br />

HiTrap columns, supplied with predefined elution conditions, a step elution using a simple<br />

syringe can be used. HiTrap columns can also be used with a chromatography system such<br />

as ÄKTAprime. The use <strong>of</strong> a chromatography system is essential when gradient elution is<br />

required.<br />

A 280<br />

A 280<br />

Elution<br />

conditions<br />

Linear<br />

change<br />

in elution<br />

conditions<br />

Binding<br />

conditions<br />

Binding<br />

conditions<br />

Time/vol.<br />

Time/vol.<br />

Fig. 6a. Step elution.<br />

Fig. 6b. Gradient elution.<br />

During development <strong>and</strong> optimization <strong>of</strong> affinity purification, use a gradient elution to scan<br />

for the optimal binding or elution conditions, as shown in Figure 7 <strong>and</strong> Figure 8.<br />

A 280 nm<br />

0.3 UV 280 nm<br />

Imidazole (M)<br />

0.5<br />

Programmed elution<br />

buffer conc.<br />

0.2<br />

0.1<br />

(His) fusion<br />

protein<br />

1 2<br />

0.4<br />

0.3<br />

0.2<br />

Sample:<br />

Column:<br />

Binding buffer:<br />

Elution buffer:<br />

Flow:<br />

System:<br />

Clarified homogenate <strong>of</strong> E. coli<br />

expressing His fusion protein<br />

HiTrap Chelating HP 1 ml column<br />

charged with Ni 2+<br />

20 mM sodium phosphate,<br />

0.5 M sodium chloride,<br />

10 mM imidazole, pH 7.4<br />

20 mM sodium phosphate,<br />

0.5 M sodium chloride,<br />

0.5 M imidazole, pH 7.4<br />

1 ml/min<br />

ÄKTAprime<br />

0<br />

0.1<br />

0<br />

0 45 65 min<br />

1: selected imidazole concentration<br />

for elution <strong>of</strong> impurities<br />

2: selected imidazole concentration<br />

for elution <strong>of</strong> pure (His) fusion protein<br />

6<br />

Fig. 7. Gradient elution <strong>of</strong> a (His) 6<br />

fusion protein.<br />

20

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