title: Affinity purification of heme-tagged proteins.
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.
authors.affiliation: Division of Molecular Therapeutics Department of Psychiatry, Columbia University, NYSPI Kolb Annex, 3rd Floor 1051 Riverside Drive, Unit 25, New York, NY, 10032, USA, wa2186@columbia.edu.
authors.forename: Wesley B
authors.initials: WB
authors.lastname: Asher
authors.forename: Kara L
authors.initials: KL
authors.lastname: Bren
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008967:Molecular Biology / Q000379:methods*; D014176:Protein Biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011993:Recombinant Fusion Proteins; C031690:heme C; D006418:Heme; D006639:Histidine