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Affinity Purification of Heme-Tagged Proteins

  • Wesley B. AsherEmail author
  • Kara L. Bren
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)

Abstract

Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an l-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay.

Key words

Affinity protein purification Affinity tag Heme-tag Visible-tag l-histidine immobilized Sepharose chromatography Protein quantification Visible tracking 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Division of Molecular Therapeutics Department of PsychiatryColumbia UniversityNew YorkUSA
  2. 2.Department of ChemistryUniversity of RochesterRochesterUSA

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