Bio-Rad Nuvia™ IMAC Resin - Manual (Page 11)

Nuvia IMAC Ni-Charged Resin 7
Section 3
General IMAC Procedures
Protein Binding
Protein adsorption to immobilized ions is performed around
neutral to slightly alkaline pH conditions (pH 7.0–8.0). To reduce
nonspecific ionic effects, concentrations of up to 1 M NaCl may
be added to the binding solution. Recombinant 6x histidine tags,
located at either the amino or carboxyl terminus of the protein, can
bind with high affinity to the matrix even when the 6x histidine tag
isn’t completely accessible. In general, the fewer the number of
accessible histidine residues, the weaker the protein binding is to
the affinity matrix. Untagged proteins that have naturally occurring
and noncontiguous histidine residues also bind to IMAC resins, but
with much lower affinity.
Batch mode binding is a good alternative if proteins are expressed
at low levels or if the overall concentration of the recombinant
6x histidine tag is low. In this case, proteins are bound to the
Nuvia
™ IMAC resin in solution prior to packing the protein-resin
complex into a liquid chromatography column for wash and elution
steps. Altering the imidazole concentration of the lysis buffer may
also optimize binding. Low concentrations (0–15 mM imidazole)
are recommended and will aid in reducing nonspecific binding of
weakly interacting proteins.
Many additives can be used without affecting the binding of
histidine-tagged proteins to IMAC resins. For example, urea,
GuHCl, nonionic detergents, and organic solvents (refer to
Section 2, Table 2) are all valid options. Chelating agents, such as
EDTA or citrate, should not be included. Reducing agents such as
β-mercaptoethanol and DTT may be used at low concentrations.
Potassium phosphate or sodium phosphate buffers are
recommended solutions for equilibration and binding.
Recommended binding buffer:
• 20–50 mM sodium or potassium phosphate, containing up to
1.0 M NaCl.
Begin with: 50 mM sodium phosphate, 0.3 M NaCl, pH 8.0