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34 Charlton and Zachariou
could also be used; however, a good chelating stationary phase to use this metal
ion in IMAC for the purification of proteins does not exist commercially. Al 3+ is
also another example, however, the commercially available 8-hydroxyquinoline
support would be more useful over IDA stationary phases for this metal ion.
Borderline Lewis metal ions like Cu 2+ can also be used in this mode (7,14).
9. Not all supports should be stored charged with metal ions. Silica-based supports
should be stored free of metal ion and only charged when required. The charged
metal ion causes a localized low pH microenvironment that can damage these
supports over time, decreasing the life expectancy of the column.
10. Under these conditions, histidine interaction with the IMCC should be quenched
(7). Furthermore, the use of oxygen-rich buffers such as phosphate, acetate,
carbonate and so on should be avoided whilst equilibrating hard Lewis IMCCs.
Sulphonic acid-based buffers such as MES and other Good’s buffers used at ≤20
mM have minimal interference and can be used.
11. Any metal ion that can be hydrolyzed can be employed with any commercially
available chelating stationary support for this section of work.
References
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chromatography, a new approach to protein fractionation. Nature 258, 598–599.
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