Immobilized metal chelate affinity chromatography (IMAC) is utilizing free coordination sites of
metal ions to bind different proteins and peptides. The principle mechanism is based on the
interaction between a metal ion coordinated to a covalently bound chelating ligand with histidine
containing proteins. For Fractogel® EMD Chelate media, iminodiacetic acid has been chosen as the
metal chelating ligand.
IDA is a very suitable ligand, because a bidentate chelating moiety remains free after
immobilization, to which a metal ion can be coordinated.
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The binding of the metal ion occurs through the nitrogen atom and two carboxylate oxygens. The metal
will coordinate 4-6 ligands, the remaining coordination sites are occupied by water which can be
displaced by proteins.
Getting Started Now (Practical hints)
Chromatographic Conditions
Since the pKa value of histidine lies in the neutral range, the binding of protein samples to the
column should normally occur at a pH value of approximately 7. Because the pKa value of an amino
acid can deviate from the theoretical pKa value, an application buffer of pH 8 often achieves
an improved binding. For optimum binding of the protein to the immobilized metal complex, the
buffer system can also have an influence. For example, Tris-containing buffers and high
concentrations of imidazole, histidine or glycine strongly influence the protein binding.
To avoid ionic interactions between proteins and carboxy groups, which might remain uncharged
with metal ions, a high ion strength (typically 0.5 M NaCl) should be present during operation.
Elution can be performed either by displacement of the protein with a competitive molecule or
by changing the pH value. In the first case, the component added to the buffer displaces the
protein from the column when an appropriate concentration is reached.
In most of the cases a gradient generated with imidazole is used. By lowering the pH value of
the buffer the binding affinity of the complex protein-metal ion is reduced and the protein
can be desorbed. The declining pH gradient can be run as a step gradient and/or as linear
gradient. Step wise gradients are often preferable, however, linear gradients may improve
the resolution.
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