Bio-Rad Nuvia™ IMAC Resin - Manual (Page 12)

8 Nuvia IMAC Ni-Charged Resin
Washes
Stringency of 6x histidine-tag binding can be effectively increased
by 1) including low concentrations of imidazole in the binding and
wash solutions, or 2) reducing the pH. Generally, highly expressed
proteins, such as those from a bacterial expression system, have
fewer contaminant proteins that copurify along with the protein of
interest. Endogenous protein contaminants are more abundant
in eukaryotic expression systems and tend to bind to the IMAC
adsorbent more weakly. In these instances, nonspecific binding
of proteins containing neighboring histidine residues becomes
a problem. These endogenous species may be washed from
the resin by either lowering the pH to 6.3 or adding imidazole to
binding and wash solutions in concentrations of 5–30 mM. The
optimal pH and/or imidazole concentration used in wash buffers
is always protein dependent and should always be determined
experimentally.
Recommended wash buffer:
• 5–30 mM imidazole; for example, 50 mM sodium phosphate,
0.3 M NaCl.
Begin with: 5 mM imidazole, 50 mM sodium phosphate,
0.3 M NaCl, pH 8.0
Elution
Desorption of the histidine-tagged protein may be accomplished in
one of three ways: introduction of a competitor ligand, reduction of
the pH, or stripping of the immobilized metal.
In competitive elution, a step or gradient elution with ligands such
as imidazole, histidine, histamine, or glycine may be carried out.
When using a gradient elution with imidazole, it is important to pre-
equilibrate the column with low concentrations of imidazole (1 mM)
and include the same concentration in the sample. This prevents
adsorption of imidazole onto the resin from triggering a drop in pH,
which might prematurely elute bound histidine-tagged proteins.
Lowering the pH of the elution buffer (pH 4.5–5.3) also releases
bound histidine-tagged proteins. In this case, the histidine residues
become protonated and are unable to bind to the immobilized ion.
Protein sensitivity to low pH ranges, however, must be taken into
consideration.