Bio-Rad Profinity IMAC Resins - Manual (Page 27)

23
Section 10
Medium-Pressure Column Purification —
Using an Imidazole Gradient to Determine
Optimal Purification of His-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 elutions may be used for subsequent purification of larger
sample volumes.
With step elutions, 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 BioLogic DuoFlow
™ system, to establish a linear
gradient.
Materials
Reagent
• Binding buffer
–
20 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 20 mM imidazole
• Elution buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 500 mM imidazole
Equipment
• Chromatography system with dual pump and gradient capability
• Fraction collector
• IMAC column (as prepared in Section 4)
Biological Sample
• Clarified lysate (as prepared in Section 7)
Note: Keep the sample as small as possible during optimization of binding and
elution conditions.
Additional Materials
• Equipment for assessing protein purity and recovery of the His-tagged
protein
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