Authors: Daniel Harder & Dimitrios Fotiadis ### Abstract 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. ### Reagents 1. Tris - NaCl - n-dodecyl-β-D-maltoside (DDM) or another detergent of choice - histidine - glycerol - NaN3 - Ni-NTA Superflow beads (Qiagen) - Wizard Midicolumn (Promega) ### Equipment - SDS-PAGE facility ### Procedure 1. 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. ### Timing When starting with solubilized membranes or membrane pellets before detergent solubilization, protein purification takes half or one day, respectively. ### References - 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 1. **Projection Structure of a Member of the Amino Acid/Polyamine/Organocation Transporter Superfamily**. F. Casagrande, M. Ratera, A. D. Schenk, M. Chami, E. Valencia, J. M. Lopez, D. Torrents, A. Engel, M. Palacin, and D. Fotiadis. *Journal of Biological Chemistry* 283 (48) 33240 - 33248 23/09/2008 [doi:10.1074/jbc.M806917200](http://dx.doi.org/10.1074/jbc.M806917200) - **Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay**. Daniel Harder and Dimitrios Fotiadis. *Nature Protocols* 7 (9) 1569 - 1578 [doi:10.1038/nprot.2012.090](http://dx.doi.org/10.1038/nprot.2012.090) ### Author information **Daniel Harder & 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 Correspondence to: Dimitrios Fotiadis (dimitrios.fotiadis@ibmm.unibe.ch) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2396) (2012) doi:10.1038/protex.2012.034. Originally published online 7 August 2012*.