Re-purification of His-tagged protein over IMAC - (Nov/21/2016 )

Hello.  Has anyone ever tried taking the elution of a his-tagged protein off of an IMAC column, buffer exchanging back into binding buffer (with the same or higher level of imidazole) and re-purifying over an IMAC column?  I'm wondering if it might be a way to increase the purity by increasing the ratio of tagged protein to host cell contaminants but have never tried it.

it will work but you may suffer unacceptable losses.

No, this will do absolutely nothing to improve purity.

Your protein will re-bind...but so will all the other contaminants that bound the first time. If contaminants binds once, they'll bind twice.

This is why expressing proteins with cleavable (solubility) tags (e.g. SUMO, GST, MBP, GFP, etc) and re-running over the column while your un-tagged protein flows through is a wonderful purification step...all the contaminants that bound the first time also bind the second time...while your tag-less protein flows through.

Alternatively, use ion exchange and/or gel filtration if you need higher purity.