Bio-Rad Nuvia™ IMAC Resin - Manual (Page 13)

Nuvia IMAC Ni-Charged Resin 9
If lower pH is used to elute bound proteins, tubes filled with a
strong neutralizing buffer such as 1 M Tris-HCl, pH 8.0 may be
used to collect acidic eluates (100–200 µl eluate/ml neutralizing
buffer). The recommended range is pH 3–5 with acetate buffers
being a preferred choice. Weakly bound contaminants may be
washed off in an intermediate wash at around pH 5.5–6.5.
Strong chelating agents such as EDTA and EGTA strip immobilized
ions from the column and cause the bound histidine-tagged
protein to elute as a protein-metal complex. This results in metal
ions appearing in the protein fractions.
Recommended elution buffer for Nuvia IMAC resin
Though a number of conditions can be used to elute the target
protein from the Nuvia resin, adjusting the concentration of
imidazole is recommended.
Recommended elution buffer:
• 20–500 mM imidazole; for example, 50 mM sodium
phosphate, 0.3 M NaCl.
Begin with: 0.5 M imidazole, 50 mM sodium phosphate,
0.3 M NaCl, pH 8.0
Purification under Denaturing Conditions
When overexpressed in E. coli, some proteins may aggregate,
forming what are known as inclusion bodies. These inclusion
bodies need to be solubilized in strong denaturants such as
6 M guanidine HCl or 8 M urea in order to purify the histidine-
tagged protein. Usually proteins expressed as inclusion bodies
are not in their native conformation, so high concentrations of
denaturants may be used during lysate preparation and protein
purification.
In order to restore the native conformation and activity of the
protein, the denaturant must be removed by dilution, dialysis, or
size exclusion chromatography. Renaturation of the protein while it
is still bound to the IMAC column is a good alternative and offers
several advantages. Aggregation may be kept to a minimum if the
protein refolds on the column when the denaturant is removed.
Higher concentrations of the refolded protein may therefore be
collected.