Bio-Rad Profinity IMAC Resins - Manual (Page 30)

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6. Wash the column with a minimum of 5 column volumes of binding or
starting buffer, containing some amount of imidazole that will not elute
the target protein.
7. Elute the His-tagged protein with 5 column volumes of elution buffer with
optimized imidazole concentration.
8. Wash the column with 5 column volumes of elution buffer. Stop
collecting fractions.
9. Reequilibrate the column with 10 column volumes of equilibration
(binding) buffer.
10. Assess the purity and recovery of the target protein.
Use an activity assay (Bio-Rad’s protein assay kit), UV absorbance,
SDS-PAGE, or western blot analysis with anti-histidine antibodies or
antibodies specific to the target protein.
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