Method Article
Purification of His-tagged membrane proteins from detergent-solubilized membranes
https://doi.org/10.1038/protex.2012.034
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posted 07 Aug, 2012
You are reading this latest protocol version
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).
The are no competing financial interests.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 07 Aug, 2012
You are reading this latest protocol version
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