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32 Charlton and Zachariou
3.3. Purification of Proteins Using IMAC in Pseudo-Cation Exchange
Mode (see Note 11)
1. Carry out steps 1–3, Subheading 3.2.
2. Equilibrate packed M n+ -IDA column with 10 CV of 30 mM MES + 30 mM
imidazole + 0.05 M NaCl pH 5.5 or 6 (see Note 10). Confirm equilibration by
measuring pH and conductivity. Continue equilibration until pH and conductivity
of effluent matches equilibration buffer.
3. Carry out steps 5–7, Subheading 3.2.
4. Subsequent wash steps can be carried out if deemed necessary (see Table 3). If
a wash step is required follow step 6, Subheading 3.2 with the appropriate wash
buffer.
5. Elute protein with up to 5 CV of 30 mM MES + 30 mM imidazole + 0.5 M
NaCl pH 5.5 or 6 at 33% of the recommended maximum linear velocity of the
stationary support, 235 cm/h for Chelating Sepharose FF. If this is insufficient to
effect elution, NaCl should be taken up to 1 M. If the target molecule still remains
Table 3
Wash type Effect Comment
Non-ionic detergents,
e.g., Triton, Tween No
more than 1% v/v
Increasing pH (>6)
Increasing ionic
strength to between
0.5Mand1M
Disrupts hydrophobic
interactions
Adjusting the pH to
beyond the isoelectric
point of the protein
will make it more
negative and interfere
with the interactions
on the adsorbent
Disrupts electrostatic
interactions
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
NaCl is used traditionally as an
eluent, however, other similar
salts could also be used