Bio-Rad Nuvia™ IMAC Resin - Manual (Page 14)

10 Nuvia IMAC Ni-Charged Resin
Finally, the use of a liquid chromatography system ensures that
the adjustment of denaturants, detergents, salts, and pH will be
effectively controlled.
Imidazole Concentrations
For optimal protein purification results, it is crucial that the
imidazole concentrations in lysis, binding, and wash buffers, as
well as elution buffers, be empirically established. Determine
optimized conditions using a small amount of sample. These
conditions may then be used to design the purification protocol
for larger samples on the same column or on a larger column. As
each protein behaves differently, it is helpful to keep the following
points in mind during lysate preparation and purification:
• Low concentrations of imidazole (1–30 mM) in lysis, binding,
and wash buffers are recommended if the potential for
background contaminants exists. The ability for nontagged
contaminating proteins to bind to the resin is generally higher
under nondenaturing conditions than under denaturing
conditions
• Low concentrations of imidazole (1–30 mM) help minimize
nonspecific binding of proteins containing noncontiguous
histidine residues by competing with them for available binding
sites on the transition metal. Competition occurs because
the imidazole ring is also found in the histidine-containing
compound
• If the recombinant histidine-tagged protein does not bind
under higher concentrations, the imidazole concentration can
be reduced for binding and wash steps
• A gradient elution test may be used to determine
concentrations of imidazole for wash and elution steps. Once
the imidazole concentration to elute the protein is established,
large samples and/or columns may be used