Isolation Purification and Separation Biochemistry

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Authors: Daniel Harder & Dimitrios Fotiadis

Abstract

This protocol describes a method to prepare detergent-solubilized membranes from Escherichia coli (E. coli), e.g. containing an overexpressed membrane protein. The procedure takes less than one day. Cells are broken by pressure cell and membranes are isolated and washed by differential centrifugation. Finally, the membranes are solubilized with the detergent of choice.

Reagents

  1. Cell pellet of E. coli
  2. Tris
  3. EDTA
  4. NaCl
  5. n-dodecyl-β-D-maltoside (DDM) or another detergent of choice glycerol
  6. NaN3

Equipment

  1. French Press
  2. ultracentrifuge

Procedure

  1. Resuspend the cell pellet from 1 l of E. coli culture in Lysis Buffer (20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA). Adapt the buffer volume to your French pressure cell.
  2. Disrupt E. coli cells by passage through a French pressure cell (20,000 psi) and remove unbroken cells by centrifugation at 10,000g (4˚C, 10 min).
  3. Ultracentrifuge the supernatant at 100,000g (4˚C, 1 h).
  4. Resuspend and homogenize the pellet containing the E. coli membranes in Lysis Buffer and ultracentrifuge again.
  5. Remove water-soluble proteins adhering to the membrane by homogenization in 20 mM Tris-HCl, pH 8.0, 300 mM NaCl and ultracentrifugation.
    • Breakpoint: Membrane pellet can be resuspended in 1 ml buffer without detergent and stored at -80 °C for months to years prior detergent solubilization.
  6. Resuspend and solubilize the membrane pellet 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).
  7. Ultracentrifuge at 100,000g (4˚C, 50 min).
  8. The supernatant represents solubilized membranes, which can be used for purification of the His-tagged protein or SPA-binding experiments directly.

Timing

The procedure takes less than one day.

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
  2. 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

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 ([email protected])

Source: Protocol Exchange (2012) doi:10.1038/protex.2012.033. Originally published online 7 August 2012.

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