Bio-Rad Profinity IMAC Resins - Manual (Page 18)

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Section 6
Immobilizing Metal Ions
Efficacy of protein binding by IMAC is dependent 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 the amino acid residues. Profinity
IMAC uses a tridentate ligand (IDA), which leaves three 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
by the choice of metal ion used, understanding 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. Profinity IMAC, based on the polymeric UNOsphere
™ technology, has
specifically been formulated with an optimal amount 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.
After slurry packing is complete (see Sections 4 and 5), the column is ready for
application of metal ions.
2. 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).
3. Apply 3–5 column volumes of the metal ion solution.
4. Wash with 5 column volumes of 50 mM sodium acetate, 0.3 M NaCl,
pH 4.0.
Remove excess ions by washing.
5. Wash with 10 column volumes of deionized water.
6. 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.
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