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

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

DOI:
10.3791/58012-v

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08:02 min

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August 23, 2018

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,
1Department of Chemistry,Muhlenberg College

Capitoli

  • 00:04Titolo
  • 00:34Preparation of the IMAC Column
  • 01:42Purification of Polyhistidine-tagged Target by IMAC
  • 04:32Reconstitution of Target Protein with Fe2+
  • 06:43Results: Undergraduate Performed Assay of a Polyhistidine Tagged Metalloenzyme
  • 07:28Conclusion

Summary

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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.

Tags

Benchtop Immobilized Metal Affinity ChromatographyPolyhistidine Tagged MetalloenzymeUndergraduate LaboratoryNon-heme Iron-binding EnzymesNickel-bound Nitriloacetic Acid ResinLysis Bind BufferCell-free Crude ExtractsFlow-throughEluent

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