Bio-rad Profinity IMAC Resins User Manual download pdf (Page 34)

3. Apply a linear gradient from 100% refolding buffer to 100% phosphate

elution buffer, with high imidazole.

Note: Elution is done by a gradient from 20 mM to 500 mM imidazole.

4. Access protein purity and recovery of fractions containing His-tagged

proteins.

Use an activity assay (Bio-Rad’s protein assay kit), SDS-PAGE, or western blot

analysis with anti-histidine antibodies or antibodies specific to the target

protein.

5. If necessary, add an additional chromatography step.

Size exclusion chromatography may be a good choice because aggregates of

un-refolded protein can be removed and the buffer composition of the purified

material can be changed simultaneously.

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Bio-rad Profinity IMAC Resins User Manual Page 34