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Immobilized Metal Ion Affinity Chromatography 33
bound, then elute with 30 mM HEPES + 30 mM imidazole +1MNaCl pH 8.
Samples should be examined on SDS–PAGE for purity (17).
6. Follow steps 10–12, Subheading 3.2.
4. Notes
1. All columns pre-charged with metal should be washed with acid to release any
loosely bound metal ions.
2. A slow loading velocity improves the diffusion of proteins (particularly, large
proteins) through pores and onto the IMCC and hence improves yields. The stated
linear velocities have been derived from the author’s personal experience and
will vary depending on the stationary support. For example, Poros supports can
have linear dynamic capacities, in some cases up to 7000 cm/h, before decreases
in capacities are observed. The maximum linear velocity of the support stated
for these methods, Chelating Sepharose FF, is 700 cm/h (18). Care must also be
taken to ensure that if prolonged loading times are chosen, the target protein is
not subject to destabilizing factors such as proteolysis or any intrinsic instability
such as deamidation or oxidation and should be monitored during the process. In
these instances, the molecule stability needs to take precedence over slow loading
velocities.
3. This amount is conservative relative to the manufacturer’s claims of 5 mg of
protein per ml Chelating Sepharose FF resin (18). However, capacities of