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146 Charlton and Zachariou
the Table, for example, increasing imidazole concentration, inclusion of amines
or other salts, and then increase the stringency of the conditions up to the highest
possible levels that do not elute the target protein.
5. Carry out steps 7–12, Subheading 3.1.
4. Notes
1. All columns pre-charged with metal should be washed with acid to release any
loosely bound metal ions.
2. This step serves to totally quench the immobilized metal ion with imidazole,
improving selectivity of the IMCC for proteins. Furthermore, it creates a uniform
surface by eluting weakly bound hydroxide species bound to the IMCC surface.
Such species have been observed previously and if not controlled can significantly
contribute to non-specific electrostatic interactions during IMAC (21). Lower
imidazole concentrations are not as effective. In addition, the pre-charge buffer
approximates the elution buffer and so can reduce metal ion leakage attributable
to such a high imidazole concentration even before elution occurs.
3. The pH of equilibration is varied throughout the literature and can range from 7
to 8. By operating closer to pH 7 than to pH 8 during protein binding, a greater
selectivity may be achieved which would ultimately yield greater purity of the final
product. Improved capacity may also result because less non-specific interactions
will occur. Most His-tagged proteins will bind within pH 7–8 range and should be
determined empirically. Other buffers such as 100 mM phosphate are commonly
used at pH 7–7.5. In these instances, the Ni-NTA becomes less selective and
proteins containing histidine regions are more likely to bind, than if imidazole
was used, leading to potential problems downstream of the process.
4. 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 and Hyper D
supports can have linear dynamic capacities, in some cases up to 7000 cm/h, before
decreases in capacities are observed. 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. The status of the protein should therefore be monitored during the
process. In these instances, the stability of the molecule needs to take precedence
over slow loading velocities.
5. This amount is conservative relative to the manufacturer’s claims of 5–10 mg of
protein per ml Ni-NTA resin (22); however, capacities of