title: Purification of His-Tagged Proteins.
abstract: Ni-NTA affinity purification of His-tagged proteins is a bind-wash-elute procedure that can be performed under native or denaturing conditions. Here, protocols for purification of His-tagged proteins under native, as well as under denaturing conditions, are given. The choice whether to purify the target protein under native or denaturing conditions depends on protein location and solubility, the accessibility of the His tag, and the desired downstream application. His-tagged proteins can be purified by a single-step affinity chromatography, namely immobilized metal ion affinity chromatography (IMAC), which is commercially available in different kinds of formats, Ni-NTA matrices being the most widely used. The provided protocols describe protein purification in the batch binding mode and apply gravity-assisted flow in disposable columns; this procedure is simple to conduct and extremely robust. IMAC purification can equally be performed in prepacked columns using FPLC or other liquid chromatography instrumentation, or using magnetic bead-based methods (Block et al., 2009).
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Spriestersbach
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Kubicek
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany. Electronic address: frank.schaefer@qiagen.com.
authors.forename: Frank
authors.initials: F
authors.lastname: Schäfer
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany.
authors.forename: Helena
authors.initials: H
authors.lastname: Block
authors.affiliation: QIAGEN GmbH, Research and Development, Qiagenstrasse 1, 40724 Hilden, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Maertens
mesh_terms: D000818:Animals; D001671:Biochemistry / Q000379:methods; D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D008280:Magnetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility
chemical_list: D002614:Chelating Agents; D011994:Recombinant Proteins; D006639:Histidine
keywords: Batch purification; Denaturing conditions; E. coli lysate; His-tagged proteins; IMAC; Ni-NTA