Home › Biochemistry › Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory
Biochemistry JoVE (Open Access) Citable · DOI

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

DOI: 10.3791/58012-v
What you'll learn
  • ✓ Perform benchtop IMAC column preparation and polyhistidine-tagged protein purification
  • ✓ Reconstitute a metalloenzyme with Fe2+ cofactor
  • ✓ Assay enzyme activity post-reconstitution using undergraduate-appropriate methods
Protocol

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Difficulty
intermediate
Total time
~4–6 hours
Biosafety
BSL-1

Steps

1
Prepare immobilized metal affinity chromatography column

Set up a benchtop IMAC column by loading resin and equilibrating with appropriate buffer. This step prepares the stationary phase for protein binding and purification.

▶ 00:34
2
Purify polyhistidine-tagged protein by IMAC

Load cell lysate containing the His-tagged dioxygenase onto the IMAC column, wash, and elute the target protein. The polyhistidine tag binds immobilized metal ions, enabling separation from contaminants.

▶ 01:42
3
Reconstitute target protein with ferrous iron

Add Fe2+ to the purified protein to restore the active metalloenzyme. This step restores catalytic function of the non-heme iron dioxygenase.

▶ 04:32
4
Assay reconstituted metalloenzyme activity

Perform enzymatic assay on the reconstituted dioxygenase to verify proper cofactor incorporation and catalytic activity. Suitable for undergraduate-level analysis.

▶ 06:43
💬 Comments coming soon