Bio-Rad Profinity IMAC Resins - Manual (Page 35)

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Section 12
Sample Preparation-Size Spin-Column
Purification of His-Tagged Proteins Using
Nondenaturing Conditions
Materials
Reagents
• Binding/wash buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 5 mM imidazole
Adjust to pH 8.0.
• Optimized wash buffer with imidazole (optional, see Section 10)
–
A wash buffer containing slightly less imidazole than necessary to
elute the target protein may be used to increase the stringency of the
wash step. Refer to Section 10, Medium-Pressure Column
Purification — Using an Imidazole Gradient to Determine Optimal
Purification of His-Tagged Proteins.
– Once the concentration of imidazole in the wash step is determined
using medium-pressure column chromatography, a stepwise elution
step may be carried out as indicated in this protocol.
• Elution buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 500 mM imidazole
Adjust to pH 8.0.
Equipment
• Sample preparation-sized IMAC spin column (as prepared in Section 5)
(for example, Micro Bio-Spin
™ columns, catalog #732-6204)
• Plasticware, 2 ml capped, 2 ml capless tubes
Biological Sample
• Clarified lysate (as prepared in Section 7)
The binding capacity of the Profinity IMAC resin is ≥15 mg His-tagged
protein per ml resin. Larger amounts of protein will require a larger
column.
Additional Materials
• Equipment for assessing protein purity and recovery of the His-tagged
protein
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