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The Use of Detergents to Purify Membrane Proteins

Thomas Arnold1,  Dirk Linke1

1Max Planck Institute for Developmental Biology, Tübingen, Germany

Publication Name: 
Current Protocols in Protein Science
Unit Number: 
Unit 4.8
DOI: 
10.1002/0471140864.ps0408s53
Online Posting Date: 
August, 2008
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Dirk Linke

Abstract

Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, or biomolecular NMR. Protocols in this unit include the isolation and solubilization of biological membranes, phase separation, and support protocols for detergent removal and detergent exchange using different methods. Curr. Protoc. Protein Sci. 53:4.8.1-4.8.30. © 2008 by John Wiley & Sons, Inc.

Keywords: detergent; membrane protein; solubilization; phase separation; cloud point; detergent removal; detergent exchange

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparative Isolation of Membrane Proteins from Escherichia coli
  • Basic Protocol 2: Purification of Membrane Proteins by Phase Separation with Triton X-114 using a Temperature Shift
  • Basic Protocol 3: Purification of Membrane Proteins by Phase Separation on C8POE Induced by an Increase in Ionic Strength
  • Detergent Removal/Detergent Exchange
  • Support Protocol 1: Detergent Removal Using Biobeads
  • Support Protocol 2: Detergent Removal or Detergent Exchange Using Dialysis
  • Alternate Protocol 1: Detergent Exchange Using Dialysis
  • Support Protocol 3: Detergent Removal Using Affinity Chromatography
  • Alternate Protocol 2: Detergent Exchange Using Affinity Chromatography
  • Support Protocol 4: Detergent Removal Using Ion Exchange
  • Alternate Protocol 3: Detergent Exchange Using Ion-Exchange Chromatography
  • Support Protocol 5: Detergent Removal Using Gel Filtration
  • Alternate Protocol 4: Detergent Exchange Using Gel Filtration
  • Support Protocol 6: Detergent Removal Using Acetone for Electrophoresis, Mass Spectrometry, or Antibody Production
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparative Isolation of Membrane Proteins from Escherichia coli

 Materials
  • Escherichia coli overnight culture
  • LB medium (see appendix 4A)
  • Resuspension buffer (see recipe)
  • CM solubilization buffer (see recipe)
  • OM solubilization buffer (see recipe)
  • Spectrophotometer, wavelength 578 nm
  • 5-liter flask
  • Culture shaker, temperature controlled
  • Ultracentrifuge
  • Magnetic stirrer
  • Additional reagents and equipment for lysing cells using a French pressure cell (unit 6.2)

Basic Protocol 2: Purification of Membrane Proteins by Phase Separation with Triton X-114 using a Temperature Shift

 Materials
  • Sucrose buffer (see recipe)
  • Isolated membranes (Basic Protocol 1)
  • Triton X-114 buffer (see recipe)
  • Microcentrifuge tubes
  • 30°C incubator
  • Laboratory centrifuge (ideally with swing-out rotor)
  • Additional reagents and equipment for checking protein content by SDS-PAGE (unit 10.1)

Basic Protocol 3: Purification of Membrane Proteins by Phase Separation on C8POE Induced by an Increase in Ionic Strength

 Materials
  • Membrane protein solution in 1% to 3% w/v C8POE (e.g., Basic Protocol 1)
  • Saturated (NH4)2SO4 solution (saturated at 4°C)
  • Glass cylinder
  • Magnetic stirrer
  • Laboratory centrifuge

Support Protocol 1: Detergent Removal Using Biobeads

 Materials
  • Protein sample (protein-detergent solution)
  • BioBeads (BioRad)
  • Methanol
  • 10 mM potassium phosphate, pH 7.2 (equilibration buffer); see appendix 2E
  • 1% (w/v) Triton X-100
  • Paper filter
  • 1 × 8–cm gravity-flow column
  • Additional reagents and equipment for performing protein assay (unit 3.4)

Support Protocol 2: Detergent Removal or Detergent Exchange Using Dialysis

 Materials
  • Protein-detergent solution with a high detergent concentration (e.g., in C8POE from Basic Protocol 3)
  • Dialysis buffer: 1% C8POE, 20 mM Tris·Cl, pH 8.0 (see appendix 2E for Tris buffer)
  • Glass beakers
  • Dialysis tubing membrane with appropriate MWCO
  • Dialysis clamps
  • Magnetic stirrer

Alternate Protocol 1: Detergent Exchange Using Dialysis

 Additional Materials (see Support Protocol 2)
  • Exchange dialysis buffer (e.g., 1% octylglucoside, 20 mM Tris·Cl, pH 8.0)

Support Protocol 3: Detergent Removal Using Affinity Chromatography

 Materials
  • Buffer A: 1% C8POE, 20 mM Tris·Cl, pH 8.0 (see appendix 2E for Tris buffer)
  • Buffer B: 1% C8POE, 500 mM imidazole, 20 mM Tris·Cl, pH 8.0
  • Ni-NTA column (unit 9.4)
  • Buffered protein (His10-tag) solution in a high concentration of C8POE (obtained using Basic Protocol 3 with His-tagged protein)
  • Sonicator bath
  • Chromatography setup with pump, UV-detector, and sample collector; alternatively, a gravity-flow column can be used
  • Additional reagents and equipment for eluting the protein using a linear gradient (unit 9.4)

Alternate Protocol 2: Detergent Exchange Using Affinity Chromatography

 Materials
  • Buffered protein (His10-tag) solution in a high concentration of C8POE (obtained using Basic Protocol 3 with His-tagged protein)
  • Buffer A: 1% C8POE, 20 mM Tris·Cl, pH 8.0 (see appendix 2E for Tris buffer)
  • Buffer X: 1% octylglucoside, 20 mM Tris·Cl, pH 8.0
  • Buffer Y: 1% octylglucoside, 500 mM imidazole, 20 mM Tris·Cl, pH 8.0
  • Ni-NTA column (unit 9.4)
  • Chromatography setup with pump, UV-detector, and sample collector; alternatively, a gravity-flow column can be used
  • Additional reagents and equipment for eluting the protein using a linear gradient (unit 9.4)

Support Protocol 4: Detergent Removal Using Ion Exchange

 Materials
  • Buffer A: 1% C8POE, 20 mM Tris·Cl, pH 8.5 (see appendix 2E for Tris buffer)
  • Buffer B: 1% C8POE, 1 M NaCl, 20 mM Tris·Cl, pH 8.5
  • Anion-exchange column (unit 8.2)
  • Protein solution (essentially salt-free) in a high concentration of C8POE, 20 mM Tris·Cl, pH 8.5 (e.g., obtained by solubilizing membranes from Basic Protocol 1 with 3% C8POE)
  • Chromatography setup with pump, UV-detector, and sample collector; alternatively, a gravity-flow column can be used
  • Sonicator bath
  • Additional reagents and equipment for eluting the protein using a linear gradient (unit 8.2)

Alternate Protocol 3: Detergent Exchange Using Ion-Exchange Chromatography

 Materials
  • Buffer A; 1% C8POE, 20 mM Tris·Cl, pH 8.5 (see appendix 2E for Tris buffer)
  • Buffer X: 1% octylglucoside, 20 mM Tris·Cl, pH 8.5
  • Buffer Y: 1% octylglucoside, 1 M NaCl, 20 mM Tris·Cl, pH 8.5
  • Anion-exchange column (unit 8.2)
  • Protein solution (essentially salt-free) in a high concentration of C8POE, 20 mM Tris·Cl, pH 8.5
  • Chromatography setup with pump, UV-detector, and sample collector; alternatively, a gravity-flow column can be used
  • Sonicator bath
  • Additional reagents and equipment for eluting the protein using a linear gradient (unit 8.2)

Support Protocol 5: Detergent Removal Using Gel Filtration

 Materials
  • Protein solution in a high concentration of C8POE (e.g., solubilized bacterial outer membranes from Basic Protocol 1)
  • Gel-filtration buffer: 1% C8POE, 20 mM Tris·Cl, pH 8.0 (see appendix 2E for Tris buffer)
  • Sonicator
  • Centrifuge concentrator (e.g., Amicon concentrators from Millipore)
  • Gel-filtration column (unit 8.3)
  • Chromatography setup with pump, UV-detector, and sample collector

Alternate Protocol 4: Detergent Exchange Using Gel Filtration

 Materials
  • Protein solution in a high concentration of C8POE (e.g., solubilized bacterial outer membranes from Basic Protocol 1)
  • Detergent exchange buffer: 1% octylglucoside, 20 mM Tris·Cl, pH 8.0 (see appendix 2E for Tris buffer)
  • Sonicator
  • Centrifuge concentrator (for example Amicon concentrators from Millipore)
  • Chromatography setup with pump, UV-detector, and sample collector
  • Gel-filtration column (unit 8.3)

Support Protocol 6: Detergent Removal Using Acetone for Electrophoresis, Mass Spectrometry, or Antibody Production

 Materials
  • Any protein-detergent solution
  • Ice-cold acetone, pre-cooled in –20°C freezer
  • Vortex
  • Benchtop centrifuge (temperature controlled, at 4°C)
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Figures

  • Figure 4.8.1
    Solubilization of membrane proteins. Biological membranes are bilayers of lipid molecules. Detergents insert into the bilayer (A and B), and at high concentrations form mixed micelles with lipids and membrane proteins, rendering the proteins soluble (C).

    View Image
  • Figure 4.8.2
    Detergent phase types. Detergents exist as monomers (A), micelles (B), hexagonal phases (C), or cubic phases (D), and a number of more complex phase types depending on detergent concentration, temperature, and ionic strength of the buffer system. For the purification of membrane proteins, the micellar phase is the most relevant.

    View Image
  • Figure 4.8.3
    Sample phase diagrams. (A) Simplified phase diagram of a detergent with a lower consolute boundary. Detergents of this type are typically nonionic. (B) A simplified phase diagram of a detergent with an upper consolute boundary, which is frequently observed for zwitterionic and glycosidic detergents. Liquid crystalline phases can be hexagonal, cubic, or complex. Reproduced from Arnold and Linke (2007) with permission of Eaton Associates.

    View Image
  • Figure 4.8.4
    Chemical structures of typical nonionic detergents. Glucopyranosides and maltopyranosides typically consist of hydrocarbon chains (n = 3 to 8) linked to R-glucose or maltose with a -glycosidic bond. Oxyethyleneglycol-based detergents have the same range of hydrocarbon chains (x = 3 to 8), but are linked to polyethyleneglycol units (y = 4 to 20 or more) with an ether bond. Triton-type detergents have a branched/aromatic hydrophobic moiety and a polyethyleneglycol headgroup. Tween detergents are complex molecules with typically one fatty acid ester group (-R) and four polyethyleneglycol moieties. Digitonin is a sterol-based glycosidic detergent isolated from plants.

    View Image
  • Figure 4.8.5
    Chemical structures of typical zwitterionic detergents. CHAPS and CHAPSO are frequently used for 2D gel electrophoresis applications and differ only in a single hydroxyl group. SB and ASB detergents typically have hydrocarbon chain lengths between 8 and 16.

    View Image
  • Figure 4.8.6
    Chemical structures of ionic detergents. CTAB is a cationic detergent mostly used in DNA isolation procedures. SDS is the classic detergent for electrophoresis and cell lysis. Laurylsarcosine selectively solubilizes the inner membrane of Gram-negative bacteria. Cholate and deoxcholate are mild detergents used to form liposomes and proteoliposomes.

    View Image

Literature Cited

Literature Cited
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    Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256: 1604-1607.
    Bornsen, K.O. 2000. Influence of salts, buffers, detergents, solvents, and matrices on MALDI-MS protein analysis in complex mixtures. Methods Mol. Biol. 146: 387-404.
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    Chevallet, M., Santoni, V., Poinas, A., Rouquie, D., Fuchs, A., Kieffer, S., Rossignol, M., Lunardi, J., Garin, J., and Rabilloud, T. 1998. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 19: 1901-1909.
    Chiu, M.L., Nollert, P., Loewen, M.C., Belrhali, H., Pebay-Peyroula, E., Rosenbusch, J.P., and Landau, E.M. 2000. Crystallization in cubo: General applicability to membrane proteins. Acta Crystallogr. D Biol. Crystallogr. 56: 781-784.
    DeGrip, W.J., VanOostrum, J., and Bovee-Geurts, P.H.M. 1998. Selective detergent-extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: A novel generic approach for the preparation of proteoliposomes. Biochemical Journal 330: 667-674.
    Drummond, C.J., Warr, G.G., Grieser, F., Ninham, B.W., and Evans, D.F. 1985. Surface-Properties and Micellar Interfacial Microenvironment of N-Dodecyl Beta-D-Maltoside. Journal Of Physical Chemistry 89: 2103-2109.
    Fernandez, C. and Wüthrich, K. 2003. NMR solution structure determination of membrane proteins reconstituted in detergent micelles. FEBS Lett. 555: 144-150.
    Filip, C., Fletcher, G., Wulff, J.L., and Earhart, C.F. 1973. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol. 115: 717-722.
    Fromme, P. and Witt, H.T. 1998. Improved isolation and crystallization of Photosystem I for structural analysis. Biochimica Et Biophysica Acta-Bioenergetics 1365: 175-184.
    Gonenne, A. and Ernst, R. 1978. Solubilization of Membrane Proteins by Sulfobetaines, Novel Zwitterionic Surfactants. Analytical Biochemistry 87: 28-38.
    Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C. 1979. Properties of detergents. Methods Enzymol. 56: 734-749.
    Herbert, B. 1999. Advances in protein solubilisation for two-dimensional electrophoresis. Electrophoresis 20: 660-663.
    Hinze, W.L. and Pramauro, E. 1993. A Critical-Review Of Surfactant-Mediated Phase Separations (Cloud-Point Extractions) – Theory And Applications. Critical Reviews In Analytical Chemistry 24: 133-177.
    Hite, R.K., Raunser, S., and Walz, T. 2007. Revival of electron crystallography. Curr. Opin. Struct. Biol. 17: 389-395.
    Hitscherich, C., Jr., Aseyev, V., Wiencek, J., and Loll, P.J. 2001. Effects of PEG on detergent micelles: Implications for the crystallization of integral membrane proteins. Acta Crystallogr. D Biol. Crystallogr. 57: 1020-1029.
    Holloway, P.W. 1973. A simple procedure for removal of Triton X-100 from protein samples. Anal. Biochem. 53: 304-308.
    Hong, K. and Hubbell, W.L. 1972. Preparation and Properties of Phospholipid Bilayers Containing Rhodopsin. Proc. Natl. Acad. Sci. U.S.A. 69: 2617-2621.
    Ishihama, Y., Katayama, H., and Asakawa, N. 2000. Surfactants usable for electrospray ionization mass spectrometry. Anal. Biochem. 287: 45-54.
    le Maire, M., Champeil, P., and Moller, J.V. 2000. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim. Biophys. Acta 1508: 86-111.
    Loll, P.J., Allaman, M., and Wiencek, J. 2001. Assessing the role of detergent-detergent interactions in membrane protein crystallization. Journal Of Crystal Growth 232: 432-438.
    Loo, R.R., Dales, N., and Andrews, P.C. 1994. Surfactant effects on protein structure examined by electrospray ionization mass spectrometry. Protein Sci. 3: 1975-1983.
    Lorch, M., Fahem, S., Kaiser, C., Weber, I., Mason, A.J., Bowie, J.U., and Glaubitz, C. 2005. How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli. Chembiochem 6: 1693-1700.
    McCarthy, F.M., Burgess, S.C., Van Den Berg, B.H., Koter, M.D., and Pharr, G.T. 2005. Differential detergent fractionation for non-electrophoretic eukaryote cell proteomics. J. Proteome. Res. 4: 316-324.
    Mooney, R.A. 1988. Use of Digitonin-Permeabilized Adipocytes for Camp Studies. Methods Enzymol. 159: 193-202.
    Murray, M.G. and Thompson, W.F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321-4325.
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    Ostermeier, C. and Michel, H. 1997. Crystallization of membrane proteins. Current Opinion in Structural Biology 7: 697-701.
    Pasquali, C., Fialka, I., and Huber, L.A. 1997. Preparative two-dimensional gel electrophoresis of membrane proteins. Electrophoresis 18: 2573-2581.
    Privé, G.G. 2007. Detergents for the stabilization and crystallization of membrane proteins. Methods 41: 388-397.
    Ramsby, M.L. and Makowski, G.S. 1999. Differential detergent fractionation of eukaryotic cells. Analysis by two-dimensional gel electrophoresis. Methods Mol. Biol. 112: 53-66.
    Rigaud, J., Chami, M., Lambert, O., Levy, D., and Ranck, J. 2000. Use of detergents in two-dimensional crystallization of membrane proteins. Biochim. Biophys. Acta 1508: 112-228.
    Rigaud, J.L., Levy, D., Mosser, G., and Lambert, O. 1998. Detergent removal by non-polar polystyrene beads – Applications to membrane protein reconstitution and two-dimensional crystallization. European Biophysics Journal with Biophysics Letters 27: 305-319.
    Rosenbusch, J.P. 1974. Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding. J. Biol. Chem. 249: 8019-8029.
    Timmins, P. 2006. Detergent Binding in Membrane Protein Crystals by Neutron Crystallography. In Neutron Scattering in Biology (J. Fitter, Gutberlet, T., Katsaras, J., eds.) pp. 73-83. Springer, Berlin Heidelberg.
    Vinogradova, O., Sonnichsen, F., and Sanders, C.R., 2nd. 1998. On choosing a detergent for solution NMR studies of membrane proteins. J. Biomol. NMR 11: 381-386.
    Werck-Reichhart, D., Benveniste, I., Teutsch, H., Durst, F., and Gabriac, B. 1991. Glycerol allows low-temperature phase separation of membrane proteins solubilized in Triton X-114: Application to the purification of plant cytochromes P-450 and b5. Anal. Biochem. 197: 125-131.
    Wollmann, P., Zeth, K., Lupas, A.N., and Linke, D. 2006. Purification of the YadA membrane anchor for secondary structure analysis and crystallization. International Journal Of Biological Macromolecules 39: 3-9.
    Yeh, A.P., McMillan, A., and Stowell, M.H. 2006. Rapid and simple protein-stability screens: application to membrane proteins. Acta Crystallogr. D Biol. Crystallogr. 62: 451-457.
 Internet Resources
    http://www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html
    http://sb20.lbl.gov/cobessi/membrane_struc.htm
    http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html

The above Web sites provide data on membrane proteins of known structure, including the crystallization conditions and references.

    http://www.membranetransport.org/

TransportDB is a relational database describing the predicted cytoplasmic membrane transport protein complement for organisms whose complete genome sequence is available.

    http://www.anatrace.com/technical_info.asp
    http://www.emdbiosciences.com/html/CBC/booklets.htm

Supplier's detergent literature resources.

     
 
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Anonymous (not verified)
Posted on July 23, 2010 - 1:41am
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