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Immobilized Metal Ion Affinity Chromatography 31
Table 2
Wash type Effect Comment
Oxygen-rich buffers
such as phosphate,
glutamate, aspartate,
acetate; at 0.1 M
strength
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
Increasing pH (>6)
and/or increasing
phosphate
concentration (>0.1 M)
Eluents competing
with metal ion
for aspartate and
glutamate surface
residues
Disrupts hydrophobic
interactions
Disrupts the aspartate
and glutamate bonds
to the IMCC as well as
disrupting electrostatic
interactions if protein
has bound in mixed
mode
IMCC, Immobilized Metal Chelate Complex.
This step can also be used to
elute the target protein, so care
must be taken to select a
condition that ensures good
differentiation between
contaminants and target
protein. Acetate is the mildest
and phosphate is the strongest
eluent from this set
In particular will disrupt any
interactions between the spacer
arm and proteins as well as
protein–protein hydrophobic
interactions that may be
occurring with the target
protein. This is more effective
when applied as part of the
equilibration conditions so as to
prevent such interactions from
taking place. Inclusion of
detergent will also assist in
removing lipids or DNA (20)
This step can also be used to
elute the target protein, so care
must be taken to select a
condition that ensures good
differentiation between
contaminants and target protein
imidazole + 0.1 M K 2 HPO 4 + 0.14 M NaCl pH 8. Samples should be examined
on SDS–PAGE for purity (17).
9. After elution of the target protein, the column should be regenerated using 3 CV
of 0.2 M EDTA + 0.5 M NaCl pH 8. Washing linear velocity is not critical as
long as it does not exceed the maximum linear velocity of the stationary support
(see Note 7).
10. Wash with 10 CV of Milli Q water.
11. Wash with 5 CV of storage solution, 0.01 M NaOH, as a preservative.
12. Store column at 4°C.