Bio-Rad Nuvia™ IMAC Resin - Manual (Page 21)

Nuvia IMAC Ni-Charged Resin 17
Section 7
Medium-Pressure Column Purification
of Histidine-Tagged Proteins
For this guideline, the sample is applied to a packed column and
the proteins are eluted using a high concentration of imidazole.
The guideline does not optimize the imidazole concentration, but
instead provides for fast capture of the target protein that may be
used as a quick check for protein expression levels. Higher levels
of purity are achievable by optimizing imidazole concentrations,
which improves protein separation. See Section 8, Medium-
Column Purification – Using an Imidazole Gradient to Determine
Optimal Purification of Histidine-Tagged Proteins.
Materials
1. Reagents
Binding buffer
• 50 mM sodium phosphate (NaH
2
PO
4
)
• 300 mM NaCl
• Low concentrations imidazole* (0–15 mM)
• Adjust to pH 8.0
Wash buffer
• 50 mM sodium phosphate (NaH
2
PO
4
)
• 300 mM NaCl
• Low concentrations of imidazole* (0–30 mM)
• Adjust to pH 8.0
Elution buffer
• 50 mM sodium phosphate (NaH
2
PO
4
)
• 300 mM NaCl
• Higher concentrations of imidazole* (250–500 mM)
• Adjust to pH 8.0
* For optimal protein purification results, it is crucial that the
imidazole concentrations in lysis, binding, and wash buffers, as
well as elution buffers, be empirically established. Determine
optimized conditions using a small amount of sample.