This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Purification of His-tagged membrane proteins from detergent-solubilized membranes
Dimitrios Fotiadis
Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol describes a method to purify a His-tagged membrane protein form detergent-solubilized Escherichia coli (E. coli) membranes. Solubilized membranes are incubated with Ni-NTA that binds the His-tagged protein. After washing, His-tagged proteins are eluted with histidine. When starting with solubilized membranes or membrane pellets before detergent solubilization, protein purification takes half or one day, respectively.
Keywords
protein purification, membrane protein
Tris, NaCl, n-dodecyl-β-D-maltoside (DDM) or another detergent of choice, histidine, glycerol, NaN3, Ni-NTA Superflow beads (Qiagen), Wizard Midicolumn (Promega),
SDS-PAGE facility
Prepare solubilized membranes according to our protocol: Preparation of detergent-solubilized membranes from Escherichia coli or start with frozen membranes from 1 l culture by solubilizing in 1% DDM, 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 0.01% NaN3 for 2 h at 4˚C under gentle agitation (final volume: 7 ml) and subsequent ultracentrifugation at 100,000g (4˚C, 50 min).
Dilute the supernatant 2-fold with Washing Buffer (20 mM Tris-HCl, pH 8, 300 mM NaCl, 0.04% DDM, 5 mM histidine, 10% glycerol, 0.01% NaN3) and bind for 2 h at 4 °C to Ni-NTA Superflow beads (Qiagen; 0.5 ml bed volume).
Load the beads onto a gravity flow column (Wizard Midicolumn, Promega), wash with washing buffer (20 ml), and elute with the same buffer containing 200 mM histidine.
Collect fractions of the eluate (e.g. 250 µl fractions) and identify the fractions containing the purified target protein by SDS-PAGE.
- Purified protein can be used e.g. for SPA-binding experiments after removing the histidine by desalting columns.
When starting with solubilized membranes or membrane pellets before detergent solubilization, protein purification takes half or one day, respectively.
Casagrande, F. et al. Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily. J. Biol. Chem. 283, 33240-33248 (2008).
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CURRENT STATUS: Posted
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Posted 07 Aug, 2012
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Associated Publications
Projection Structure of a Member of the Amino Acid/Polyamine/Organocation Transporter Superfamily
by F. Casagrande, M. Ratera, A. D. Schenk, +7
Journal Of Biological Chemistry (28 May, 2012)
Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay
by Daniel Harder and Dimitrios Fotiadis
Nature Protocols (03 August, 2012)
Method Article
Purification of His-tagged membrane proteins from detergent-solubilized membranes
Dimitrios Fotiadis
Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 07 Aug, 2012
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol describes a method to purify a His-tagged membrane protein form detergent-solubilized Escherichia coli (E. coli) membranes. Solubilized membranes are incubated with Ni-NTA that binds the His-tagged protein. After washing, His-tagged proteins are eluted with histidine. When starting with solubilized membranes or membrane pellets before detergent solubilization, protein purification takes half or one day, respectively.
Tris, NaCl, n-dodecyl-β-D-maltoside (DDM) or another detergent of choice, histidine, glycerol, NaN3, Ni-NTA Superflow beads (Qiagen), Wizard Midicolumn (Promega),
SDS-PAGE facility
Prepare solubilized membranes according to our protocol: Preparation of detergent-solubilized membranes from Escherichia coli or start with frozen membranes from 1 l culture by solubilizing in 1% DDM, 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 0.01% NaN3 for 2 h at 4˚C under gentle agitation (final volume: 7 ml) and subsequent ultracentrifugation at 100,000g (4˚C, 50 min).
Dilute the supernatant 2-fold with Washing Buffer (20 mM Tris-HCl, pH 8, 300 mM NaCl, 0.04% DDM, 5 mM histidine, 10% glycerol, 0.01% NaN3) and bind for 2 h at 4 °C to Ni-NTA Superflow beads (Qiagen; 0.5 ml bed volume).
Load the beads onto a gravity flow column (Wizard Midicolumn, Promega), wash with washing buffer (20 ml), and elute with the same buffer containing 200 mM histidine.
Collect fractions of the eluate (e.g. 250 µl fractions) and identify the fractions containing the purified target protein by SDS-PAGE.
- Purified protein can be used e.g. for SPA-binding experiments after removing the histidine by desalting columns.
When starting with solubilized membranes or membrane pellets before detergent solubilization, protein purification takes half or one day, respectively.
Casagrande, F. et al. Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily. J. Biol. Chem. 283, 33240-33248 (2008).
Associated Publications
Projection Structure of a Member of the Amino Acid/Polyamine/Organocation Transporter Superfamily
by F. Casagrande, M. Ratera, A. D. Schenk, +7
Journal Of Biological Chemistry (28 May, 2012)
Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay
by Daniel Harder and Dimitrios Fotiadis
Nature Protocols (03 August, 2012)