Bio-Rad Profinity IMAC Resins - Manual (Page 11)

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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.
Finally, the use of a liquid chromatography system ensures that the adjustment of
denaturants, detergents, salts, and pH will be effectively controlled.
Sections 8 and 11 discuss preparation of lysates and purification of His-tagged
protein using denaturants.
Purification Under Nondenaturing Conditions
Purification under nondenaturing conditions might be preferred to denaturing
conditions in instances when restoration of a protein’s structure and activity are
difficult to achieve. However, the potential to bind background contaminants might
be higher under nondenaturing conditions. In these instances, a number of
parameters may be optimized to improve purification results. The addition of
nonionic detergents or glycerol might improve recovery by reducing nonspecific
hydrophobic interactions. Additionally, low amounts of imidazole in the lysis and
wash buffers are suggested to minimize levels of contaminating proteins that bind
to the IMAC adsorbent. Finally, ensuring that all buffers have sufficient ionic
strength, with the addition of NaCl, will minimize nonspecific electrostatic
interactions.
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
determined. Optimized conditions should be determined using a small amount of
sample and a 1 ml IMAC column. 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
elements in mind during lysate preparation and purification:
• Low concentrations of imidazole (0–20 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 (0–20 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 found also in the histidine-containing compound
• If binding of the recombinant His-tagged protein does not occur under higher
concentrations, the imidazole concentration can be reduced
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