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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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