Bio-Rad Nuvia™ IMAC Resin - Manual (Page 29)

Nuvia IMAC Ni-Charged Resin 25
Section 9
Sample Preparation–Sized Spin-Column Purification
of Histidine-Tagged Proteins
Materials
1. 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 8)
• 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 8, Medium-Pressure Column Purification —
Using an Imidazole Gradient to Determine Optimal
Purification of Histidine-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
Note: If necessary, up to 8 M urea may be added to these
buffers in order to solubilize proteins in inclusion bodies. See
Section 7 for more information.
2. Equipment
• Sample preparation–sized IMAC spin column (as prepared
in Section 5) (for example, Micro Bio-Spin ™ columns, cat.
#732-6204)
• Plasticware, 2 ml capped and 2 ml capless tubes