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Immobilized Metal Ion Affinity Chromatography 29
33% the maximum operating linear velocity allowed by the stationary support
(see Note 2), that is, 70–235 cm/h for the stated support. Assume a loading of
no more than 1 mg target protein per ml of stationary support (see Note 3).
However, target proteins in ratio volumes of 300:1 cell culture per support have
been successfully loaded by the author (see Note 4).
7. Wash stationary support with 10 CV of equilibration buffer at the loading linear
velocity or until the A 280 nm reading is at baseline (see Note 5).
8. Subsequent wash steps can be carried out if deemed necessary (see Table 1). If
a wash step is required follow step 7 with the appropriate wash buffer.
Table 1
Wash type Effect Comment
Glycine, Arginine,
∼0.5MNH 4 Cl and
pH 7
Non-amine salts, e.g.,
∼0.5M–1MNaCl;
in 20 mM Imidazole
+ 50 mM NaCl pH 7
Non-ionic detergents,
e.g., Triton, Tween
No more than 1% v/v
Chaotropic agents,
e.g., 4 M Urea, e.g., 4
M Guanidine–HCl
Decreasing pH
(20
mM)
Mild eluents that
compete for Ni with
histidine
Will disrupt any
non-specific
electrostatic
interactions
Disrupts hydrophobic
interactions
Disrupts the histidine
bond to the IMCC
IMCC, Immobilized Metal Chelate Complex.
These are mild eluents that will
not elute the His-tag protein but
may displace weaker bound
proteins
Such interactions are common in
IMAC particularly if the
equilibration and wash steps had