Bio-Rad Profinity IMAC Resins - Manual (Page 25)

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Section 9
Medium-Pressure Column Purification
of Histidine-Tagged (His-Tagged) Proteins
Using Nondenaturing Conditions
For this guideline, the sample is applied to a packed column and the proteins are
eluted using a high imidazole concentration. The guideline does not optimize the
imidazole concentration, but instead provides for fast capture of the target protein
and may be used as a quick check for protein expression levels.
Higher levels of purity are achievable by optimizing imidazole concentrations, which
improve protein separation. See Section 10, Medium-Column Purification – Using
an Imidazole Gradient to Determine Optimal Purification of His-Tagged Proteins.
Materials
Reagents
• Binding buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– Low concentrations imidazole* (such as 0–10 mM)
Adjust to pH 8.0.
• Wash buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– Low concentrations of imidazole* (such as 0–20 mM)
Adjust to pH 8.0.
• Elution buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– Higher concentrations of imidazole* (such as 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 as well as elution buffers be
empirically determined. Optimized conditions should be determined
using a small amount of sample and a 1 ml IMAC column.
Equipment
• IMAC column (as prepared in Section 4)
Biological Sample
• Clarified lysate (as prepared in Section 7)
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