Bio-Rad Nuvia™ IMAC Resin - Manual (Page 20)

16 Nuvia IMAC Ni-Charged Resin
Section 6
Immobilizing Metal Ions
Efficacy of protein binding by IMAC depends on two factors: the
number of available histidine, cysteine, and tryptophan residues
on a protein’s surface, and the number of coordination sites on
the immobilized ion that are not occupied by the chelating ligand
and thus available to bind amino acid residues. Nuvia
™ IMAC resin
uses a quadridentate ligand (NTA), which leaves two of the six
coordination sites on the nickel ion accessible to the protein of
interest.
Although the most commonly used ion is Ni 2+ , protein selectivity
may be increased through the choice of metal ion used,
understanding of the structure of the metal-chelate complex
and its interaction with the protein, knowledge of the protein’s
expression level, and the ligand density of the IMAC adsorbent.
While high ligand density usually means higher binding capacity, it
can also translate into lower target protein selectivity. Nuvia IMAC
resin, based on the polymeric UNOsphere
™ technology, has been
specifically formulated with an optimal number of chelating ligands
on the resin’s surface and pores to deliver both good capacity and
excellent protein purity.
1. Equilibrate the column with 5 column volumes of 50 mM
sodium acetate, 0.3 M NaCl, pH 4.0.
2. After slurry packing is complete (see Sections 4 and/or 5), the
column is ready for the removal or addition of metal ions.
3. If necessary, strip any metal ions by washing with 10 column
volumes of 50 mM sodium phosphate, 0.3 M NaCl, and
0.05–0.5 M EDTA, pH 7.5.
4. Make a 0.1–0.3 M solution of the metal ion of choice. For best
results, the pH of the solution should be <7 (neutral to weakly
acidic).
5. Apply 3–5 column volumes of the metal ion solution.
6. Wash with 5 column volumes of 50 mM sodium acetate, 0.3 M
NaCl, pH 4.0. Remove excess ions by washing.
7. Wash with 10 column volumes of deionized water.
8. Equilibrate with at least 5 column volumes of starting buffer; for
example, 50 mM sodium phosphate, 0.3 M NaCl, pH 7–8. The
column is now ready for separation.