Bio-Rad Nuvia™ IMAC Resin - Manual (Page 25)

Nuvia IMAC Ni-Charged Resin 21
Section 8
Medium-Pressure Column Purification — Using
an Imidazole Gradient to Determine Optimal
Purification of Histidine-Tagged Proteins
Gradient elution tests are useful because they do not require
optimization of imidazole concentrations, but instead may be
used to determine suitable imidazole concentrations for wash and
elution steps. Once a suitable concentration has been determined
using a gradient elution such as the one outlined below, often an
easier protocol using step elution may be used for subsequent
purification of larger sample volumes.
With step elution, the protein can be collected in smaller
volumes and at higher concentrations. Using this protocol, the
concentration of imidazole that elutes the target protein may be
calculated and used for a step protocol.
This protocol requires the use of a gradient mixer coupled to a
chromatography system such as Bio-Rad’s NGC
™ system to
establish a linear gradient.
Materials
1. Reagents
Binding buffer
• 20 mM sodium phosphate (NaH
2
PO
4
)
• 300 mM NaCl
• 20 mM imidazole
• Adjust to pH 8.0
Elution buffer
• 50 mM sodium phosphate (NaH
2
PO
4
)
• 300 mM NaCl
• 500 mM imidazole
• Adjust to pH 8.0
2. Equipment
• Chromatography system with dual pump and gradient
capability
• Fraction collector
• IMAC column (as prepared in Section 4)