Affinity Chromatography Handbook

20
Gradient and step elution
Figure 6 shows examples of step and gradient elution conditions. For prepacked affinity
HiTrap columns, supplied with predefined elution conditions, a step elution using a simple
syringe can be used. HiTrap columns can also be used with a chromatography system such
as ÄKTAprime. The use of a chromatography system is essential when gradient elution is
required.
A 280
A 280 nm
0.3
0.2
0.1
0
Binding
conditions
Fig. 6a. Step elution.
Imidazole (M)
UV 280 nm
0.5
Programmed elution
buffer conc.
(His) fusion
protein
6
0 45 65 min
1
Elution
conditions
2
Time/vol.
0.4
0.3
0.2
0.1
0
A 280
Binding
conditions
Fig. 6b. Gradient elution.
Sample: Clarified homogenate of E. coli
expressing His fusion protein
Column: HiTrap Chelating HP 1 ml column
charged with Ni 2+
Binding buffer: 20 mM sodium phosphate,
0.5 M sodium chloride,
10 mM imidazole, pH 7.4
Elution buffer: 20 mM sodium phosphate,
0.5 M sodium chloride,
0.5 M imidazole, pH 7.4
Flow: 1 ml/min
System: ÄKTAprime
1: selected imidazole concentration
for elution of impurities
2: selected imidazole concentration
for elution of pure (His) fusion protein
6
Linear
change
in elution
conditions
Time/vol.
During development and optimization of affinity purification, use a gradient elution to
scan for the optimal binding or elution conditions, as shown in Figure 7 and Figure 8.
Fig. 7. Gradient elution of a (His) 6 fusion protein.