The Use of Detergents to Purify Membrane Proteins

Marcella Orwick‐Rydmark1, Thomas Arnold2, Dirk Linke1

1 University of Oslo, Department of Biosciences, Oslo, 2 Boehringer‐Ingelheim Veterinary Research Center, Hannover
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 4.8
DOI:  10.1002/0471140864.ps0408s84
Online Posting Date:  April, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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, biomolecular NMR, or electron microscopy. A brief summary of alternative membrane protein solubilizing and stabilizing systems is also included. Protocols in this unit include the isolation and solubilization of biological membranes and phase separation; support protocols for detergent removal, detergent exchange, and the determination of critical micelle concentration using different methods are also included. © 2016 by John Wiley & Sons, Inc.

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • 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
  • Basic Protocol 4: Determination of Detergent Critical Micellar Concentration using Pyrene Fluorescence
  • 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
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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 (cytoplasmic membrane) solubilization buffer (see recipe)
  • OM (outer membrane) 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; Wingfield, )

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 ( 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; Gallagher, )

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

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

Basic Protocol 4: Determination of Detergent Critical Micellar Concentration using Pyrene Fluorescence

  Materials
  • Stock of, e.g., 10% (w/v) detergent in buffer containing 2 μM pyrene
  • Stock solution of detergent in buffer without 2 μM pyrene
  • Buffer (same as that used for the detergent) with 2 μM pyrene
  • Buffer (same as that used for the detergent) without pyrene
  • 96‐well plate if fluorimeter has this capability, or alternatively, cuvettes.
  • Fluorimeter (temperature control is desirable)
  • Software such as Origin for data analysis (recommended: Origin)

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; Olson and Markwell, )

Support Protocol 2: Detergent Removal or Detergent Exchange Using Dialysis

  Materials
  • Protein‐detergent solution with a high detergent concentration (e.g., in C8POE from 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 MWCO below the size of the protein:detergent complex
  • Dialysis clamps
  • Magnetic stirrer

Alternate Protocol 1: Detergent Exchange Using Dialysis

  Additional Materials (also see protocol 6)
  • Exchange dialysis buffer (e.g., 1% octylglucoside w/v, 20 mM Tris·Cl, pH 8.0)

Support Protocol 3: Detergent Removal Using Affinity Chromatography

  Materials
  • Buffer A: 1% C8POE w/v, 20 mM Tris·Cl, pH 8.0 (see appendix 2E for Tris buffer)
  • Buffer B: 1% C8POE w/v, 500 mM imidazole, 20 mM Tris·Cl, pH 8.0
  • Ni‐NTA column (unit 9.4; Petty, )
  • Buffered protein (His 10‐tag) solution in a high concentration of C8POE (obtained using 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; Petty, )

Alternate Protocol 2: Detergent Exchange Using Affinity Chromatography

  Materials
  • Buffered protein (His 10‐tag) solution in a high concentration of C8POE (obtained using protocol 3 with His‐tagged protein)
  • Buffer A: 1% w/v C8POE , 20 mM Tris·Cl, pH 8.0 (see appendix 2E for Tris buffer)
  • Buffer X: 1% w/v octylglucoside, 20 mM Tris·Cl, pH 8.0
  • Buffer Y: 1% w/v 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; Petty, )

Support Protocol 4: Detergent Removal Using Ion Exchange

  Materials
  • Buffer A: 1% w/v C8POE, 20 mM Tris·Cl, pH 8.5 (see appendix 22 for Tris buffer)
  • Buffer B: 1% w/v C8POE, 1 M NaCl, 20 mM Tris·Cl, pH 8.5
  • Anion‐exchange column (unit 8.2; Williams and Frasca, )
  • 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 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; Williams and Frasca, )

Alternate Protocol 3: Detergent Exchange Using Ion‐Exchange Chromatography

  Materials
  • Buffer A; 1% w/v C8POE, 20 mM Tris·Cl, pH 8.5 (see appendix 2E for Tris buffer)
  • Buffer X: 1% w/v octylglucoside, 20 mM Tris·Cl, pH 8.5
  • Buffer Y: 1% w/v octylglucoside, 1 M NaCl, 20 mM Tris·Cl, pH 8.5
  • Anion‐exchange column (unit 8.2; Williams and Frasca, )
  • 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; Williams and Frasca, )

Support Protocol 5: Detergent Removal Using Gel Filtration

  Materials
  • Protein solution in a high concentration of C8POE (e.g., solubilized bacterial outer membranes from protocol 1)
  • Gel‐filtration buffer: 1% w/v 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; Hagel, )
  • 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 protocol 1)
  • Detergent exchange buffer: 1% w/v 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; Hagel, )

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)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Aguiar, J., Carpena, P., Molina‐Bolivar, J.A., and Ruiz, C.C. 2003. On the determination of the critical micelle concentration by the pyrene 1: 3 ratio method. J. Colloid. Interface Sci. 258:116‐122. doi:10.1016/S0021‐9797(02)00082‐6.
  Ananthapadmanabhan, K.P., Goddard, E.D., Turro, N.J., and Kuo, P.L. 1985. Fluorescence probes for critical micelle concentration. Langmuir 1:352‐355. doi: 10.1021/la00063a015.
  Arnold, T. and Linke, D. 2007. Phase separation in the isolation and purification of membrane proteins. Biotechniques 43:427‐430, 432, 434 passim.
  Ashani, Y. and Catravas, G.N. 1980. Highly reactive impurities in Triton X‐100 and Brij 35: Partial characterization and removal. Anal. Biochem. 109:55‐62. doi:10.1016/0003‐2697(80)90009‐3.
  Ayorinde, F.O., Gelain, S.V., Johnson, J.H., Jr., and Wan, L.W. 2000. Analysis of some commercial polysorbate formulations using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Rapid Commun. Mass Spectrom. 14:2116‐2124. doi: 10.1002/1097‐0231(20001130)14:22<2116:AID‐RCM142>3.0.CO;2‐1.
  Barnard, T.J., Wally, J.L., and Buchanan, S.K. 2007. Crystallization of integral membrane proteins. Curr. Protoc. Protein Sci. 47:17.9.1‐17.9.15.
  Belnap, D.M. 2015. Electron microscopy and image processing: Essential tools for structural analysis of macromolecules. Curr. Protoc. Protein Sci. 82:17.2.1‐17.2.61. doi: 10.1002/0471140864.ps1702s82.
  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.
  Breukels, V., Konijnenberg, A., Nabuurs, S.M., Doreleijers, J.F., Kovalevskaya, N.V., and Vuister, G.W. 2011. Overview on the use of NMR to examine protein structure. Curr. Protoc. Protein Sci. 64:17.5.1‐17.5.44.
  Brown, D.A. and London, E. 1997. Structure of detergent‐resistant membrane domains: Does phase separation occur in biological membranes? Biochem. Biophys. Res. Commun. 240:1‐7.
  Chae, P.S., Wander, M.J., Cho, K.H., Laible, P.D., and Gellman, S.H. 2013. Carbohydrate‐containing Triton X‐100 analogues for membrane protein solubilization and stabilization. Mol. Biosyst. 9:626‐629. doi: 10.1039/c3mb25584k.
  Chae, P.S., Bae, H.E., Ehsan, M., Hussain, H., and Kim, J.W. 2014a. New ganglio‐tripod amphiphiles (TPAs) for membrane protein solubilization and stabilization: Implications for detergent structure‐property relationships. Org. Biomol. Chem. 12:8480‐8487. doi: 10.1039/C4OB01375A.
  Chae, P.S., Cho, K.H., Wander, M.J., Bae, H.E., Gellman, S.H., and Laible, P.D. 2014b. Hydrophobic variants of ganglio‐tripod amphiphiles for membrane protein manipulation. Biochim. Biophys. Acta 1838:278‐286. doi: 10.1016/j.bbamem.2013.09.011.
  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. 1998. Selective detergent‐extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: A novel generic approach for the preparation of proteoliposomes. Biochem. J. 330:667‐674.
  del Castillo, J.L., Czapkiewicz, J., Perez, A.G., and Rodriguez, J.R. 2000. Micellization of decyldimethylbenzylammonium chloride at various temperatures studied by densitometry and conductivity. Colloids Surf. A Physicochem. Eng. Asp. 166:161‐169. doi:10.1016/S0927‐7757(99)00515‐4.
  Drelich, J., Fang, C., and White, C.L. 2006. Measurement of interfacial tension in Fluid‐Fluid Systems. In Encyclopedia of Surface and Colloid Science. (P. Somasundaran, eds.), volume 4, pp. 2966‐2980. Marcel Dekker, Roca Baton, Fla.
  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. J. Phys. Chem. 89:2103‐2109. doi: 10.1021/j100256a060.
  Duquesne, K. and Sturgis, J.N. 2010. Membrane protein solubilization. Methods Mol. Biol. 601:205‐217. doi: 10.1007/978‐1‐60761‐344‐2_13.
  Fernandez, C. and Wuthrich, K. 2003. NMR solution structure determination of membrane proteins reconstituted in detergent micelles. FEBS Lett. 555:144‐150. doi: 10.1016/S0014‐5793(03)01155‐4.
  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.
  Friedmann, D., Messick, T. and Marmorstein, R. 2011. Crystallization of macromolecules. Curr. Protoc. Protein Sci. 66:17.4.1‐17.4.26.
  Fromme, P. and Witt, H.T. 1998. Improved isolation and crystallization of Photosystem I for structural analysis. Biochim. Biophys. Acta Bioenerg. 1365:175‐184. doi:10.1016/S0005‐2728(98)00059‐0.
  Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Protein Sci. 68:10.1.1‐10.1.44.
  Ghosh, E., Kumari, P., Jaiman, D., and Shukla, A.K. 2015. Methodological advances: The unsung heroes of the GPCR structural revolution. Nat. Rev. Mol. Cell Biol. 16:69‐81. doi: 10.1038/nrm3933.
  Gonenne, A. and Ernst, R. 1978. Solubilization of membrane proteins by sulfobetaines, novel zwitterionic surfactants. Anal. Biochem. 87:28‐38. doi: 10.1016/0003‐2697(78)90565‐1.
  Hagel, L. 1998. Gel‐filtration chromatography. Curr. Protoc. Protein Sci. 14:8.3.1‐8.3.30.
  Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C. 1979. Properties of detergents. Methods Enzymol. 56:734‐749. doi: 10.1016/0076‐6879(79)56066‐2.
  Herbert, B. 1999. Advances in protein solubilisation for two‐dimensional electrophoresis. Electrophoresis 20:660‐663. doi: 10.1002/(SICI)1522‐2683(19990101)20:4/5%3c660::AID‐ELPS660%3e3.0.CO;2‐Q.
  Hinze, W.L. and Pramauro, E. 1993. A critical‐review of surfactant‐mediated phase separations cloud‐point extractions—Theory and applications. Crit. Rev. Anal. Chem. 24:133‐177. doi: 10.1080/10408349308048821.
  Hite, R.K., Raunser, S., and Walz, T. 2007. Revival of electron crystallography. Curr. Opin. Struct. Biol. 17:389‐395. doi: 10.1016/j.sbi.2007.06.006.
  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. doi: 10.1107/S0907444901006242.
  Holloway, P.W. 1973. A simple procedure for removal of Triton X‐100 from protein samples. Anal. Biochem. 53:304‐308. doi: 10.1016/0003‐2697(73)90436‐3.
  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. doi: 10.1073/pnas.69.9.2617.
  Ishihama, Y., Katayama, H., and Asakawa, N. 2000. Surfactants usable for electrospray ionization mass spectrometry. Anal. Biochem. 287:45‐54. doi: 10.1006/abio.2000.4836.
  Le Bon, C., Popot, J.L., and Giusti, F. 2014. Labeling and functionalizing amphipols for biological applications. J. Membr. Biol. 247:797‐814. doi: 10.1007/s00232‐014‐9655‐y.
  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. doi: 10.1016/S0304‐4157(00)00010‐1.
  Linke, D. 2009. Detergents: An overview. Methods Enzymol. 463:603‐617. doi: 10.1016/S0076‐6879(09)63034‐2.
  Linke, D. 2014. Explanatory chapter: Choosing the right detergent. Methods Enzymol. 541:141‐148. doi: 10.1016/B978‐0‐12‐420119‐4.00011‐2.
  Loll, P.J., Allaman, M., and Wiencek, J. 2001. Assessing the role of detergent–detergent interactions in membrane protein crystallization. J. Cryst. Growth 232:432‐438. doi: 10.1016/S0022‐0248(01)01076‐4.
  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. doi: 10.1002/pro.5560031109.
  Mach, H., Middaugh, C.R., and Denslow, N. 1995. Determining the identity and purity of recombinant proteins by UV absorption spectroscopy. Curr. Protoc. Protein Sci. 1:7.2.1‐7.2.21.
  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. doi: 10.1021/pr049842d.
  McGregor, C.L., Chen, L., Pomroy, N.C., Hwang, P., Go, S., Chakrabartty, A., and Prive, G.G. 2003. Lipopeptide detergents designed for the structural study of membrane proteins. Nat. Biotechnol. 21:171‐176. doi: 10.1038/nbt776.
  Mooney, R.A. 1988. Use of digitonin‐permeabilized adipocytes for cAMP studies. Methods Enzymol. 159:193‐202. doi: 10.1016/0076‐6879(88)59020‐1.
  Murray, M.G. and Thompson, W.F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8:4321‐4325. doi: 10.1093/nar/8.19.4321.
  Neugebauer, J.M. 1990. Detergents: An overview. Methods Enzymol. 182:239‐253. doi: 10.1016/0076‐6879(90)82020‐3.
  Newstead, S., Ferrandon, S., and Iwata, S. 2008a. Rationalizing alpha‐helical membrane protein crystallization. Protein Sci. 17:466‐472. doi: 10.1110/ps.073263108.
  Newstead, S., Hobbs, J., Jordan, D., Carpenter, E.P., and Iwata, S. 2008b. Insights into outer membrane protein crystallization. Mol. Membr. Biol. 25:631‐638. doi: 10.1080/09687680802526574.
  Norris, J.L., Porter, N.A., and Caprioli, R.M. 2003. Mass spectrometry of intracellular and membrane proteins using cleavable detergents. Anal. Chem. 75:6642‐6647. doi: 10.1021/ac034802z.
  Olson, B.J. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. 48:3.4.1‐3.4.29.
  Orwick, M.C., Judge, P.J., Procek, J., Lindholm, L., Graziadei, A., Engel, A., Grobner, G., and Watts, A. 2012. Detergent‐free formation and physicochemical characterization of nanosized lipid‐polymer complexes: Lipodisq. Angew. Chem. Int. Ed. Engl. 51:4653‐4657. doi: 10.1002/anie.201201355.
  Orwick‐Rydmark, M., Lovett, J.E., Graziadei, A., Lindholm, L., Hicks, M.R., and Watts, A. 2012. Detergent‐free incorporation of a seven‐transmembrane receptor protein into nanosized bilayer Lipodisq particles for functional and biophysical studies. Nano Lett. 12:4687‐4692. doi: 10.1021/nl3020395.
  Osborn, M.J. and Munson, R. 1974. Separation of the inner cytoplasmic) and outer membranes of Gram‐negative bacteria. Methods Enzymol. 31:642‐653. doi: 10.1016/0076‐6879(74)31070‐1.
  Ostermeier, C. and Michel, H. 1997. Crystallization of membrane proteins. Curr. Opin. Struct. Biol. 7:697‐701. doi: 10.1016/S0959‐440X(97)80080‐2.
  Pain, R.H 2005a. Determining the CD spectrum of a protein. Curr. Protoc. Protein Sci. 38:7.6.1‐7.6.24.
  Pain, R.H. 2005b. Determining the fluorescence spectrum of a protein. Curr. Protoc. Protein Sci. 38:7.7.1‐7.7.20.
  Pasquali, C., Fialka, I., and Huber, L.A. 1997. Preparative two‐dimensional gel electrophoresis of membrane proteins. Electrophoresis 18:2573‐2581. doi: 10.1002/elps.1150181413.
  Petty, K.J. 1996. Metal‐chelate affinity chromatography. Curr. Protoc. Protein Sci. 4:9.9.4.1‐9.4.16.
  Phillips, A.T. and Signs, M.W. 2005. Desalting, concentration, and buffer exchange by dialysis and ultrafiltration. Curr. Protoc. Protein Sci. 38:4.4.1‐4.4.15.
  Poget, S.F. and Girvin, M.E. 2007. Solution NMR of membrane proteins in bilayer mimics: Small is beautiful, but sometimes bigger is better. Biochim. Biophys. Acta 1768:3098‐3106. doi: 10.1016/j.bbamem.2007.09.006.
  Prive, G.G. 2007. Detergents for the stabilization and crystallization of membrane proteins. Methods 41:388‐397. doi: 10.1016/j.ymeth.2007.01.007.
  Prive, G.G. 2009. Lipopeptide detergents for membrane protein studies. Curr. Opin. Struct. Biol. 19:379‐385. doi: 10.1016/j.sbi.2009.07.008.
  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.
  Raschle, T., Hiller, S., Etzkorn, M., and Wagner, G. 2010. Nonmicellar systems for solution NMR spectroscopy of membrane proteins. Curr. Opin. Struct. Biol. 20:471‐479. doi: 10.1016/j.sbi.2010.05.006.
  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. Eur. Biophys. J. Biophys. Lett. 27:305‐319. doi: 10.1007/s002490050138.
  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 1508:112‐128. doi: 10.1016/S0005‐2736(00)00307‐2.
  Rigaud, J.L., Mosser, G., Lacapere, J.J., Olofsson, A., Levy, D., and Ranck, J.L. 1997. Bio‐Beads: An efficient strategy for two‐dimensional crystallization of membrane proteins. J. Struct. Biol. 118:226‐235. doi: 10.1006/jsbi.1997.3848.
  Ritchie, T.K., Grinkova, Y.V., Bayburt, T.H., Denisov, I.G., Zolnerciks, J.K., Atkins, W.M., and Sligar, S.G. 2009. Chapter 11: Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464:211‐231. doi: 10.1016/S0076‐6879(09)64011‐8.
  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.
  Shahid, S.A., Bardiaux, B., Franks, W.T., Krabben, L., Habeck, M., van Rossum, B.J., and Linke, D. 2012. Membrane‐protein structure determination by solid‐state NMR spectroscopy of microcrystals. Nat. Methods 9:1212‐1217. doi: 10.1038/nmeth.2248.
  Thein, M., Sauer, G., Paramasivam, N., Grin, I., and Linke, D. 2010. Efficient subfractionation of gram‐negative bacteria for proteomics studies. J. Proteome Res. 9:6135‐6147. doi: 10.1021/pr1002438.
  Theisen, M.J., Potocky, T.B., McQuade, D.T., Gellman, S.H., and Chiu, M.L. 2005. Crystallization of bacteriorhodopsin solubilized by a tripod amphiphile. Biochim. Biophys. Acta 1751:213‐216. doi: 10.1016/j.bbapap.2005.04.011.
  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.
  Ujwal, R. and Bowie, J.U. 2011. Crystallizing membrane proteins using lipidic bicelles. Methods 55:337‐341. doi: 10.1016/j.ymeth.2011.09.020.
  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. doi: 10.1023/A:1008289624496.
  Vuckovic, D., Dagley, L.F., Purcell, A.W., and Emili, A. 2013. Membrane proteomics by high performance liquid chromatography‐tandem mass spectrometry: Analytical approaches and challenges. Proteomics 13:404‐423. doi: 10.1002/pmic.201200340.
  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. doi: 10.1016/0003‐2697(91)90367‐3.
  Williams, A. and Frasca, V. 1999. Ion‐exchange chromatography. Curr. Protoc. Protein Sci. 15:8.2.1‐8.2.30.
  Wingfield, P.T. 2014. Preparation of soluble proteins from Escherichia coli. Curr. Protoc. Protein Sci. 78:6.2.1‐6.2.22. doi: 10.1002/0471140864.ps0602s78.
  Wollmann, P., Zeth, K., Lupas, A.N., and Linke, D. 2006. Purification of the YadA membrane anchor for secondary structure analysis and crystallization. Int. J. Biol. Macromol. 39:3‐9. doi: 10.1016/j.ijbiomac.2005.11.009.
  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. doi: 10.1107/S0907444906005233.
  Yu, S.M., McQuade, D.T., Quinn, M.A., Hackenberger, C.P., Krebs, M.P., Polans, A.S., and Gellman, S.H. 2000. An improved tripod amphiphile for membrane protein solubilization. Protein Sci. 9:2518‐2527. doi: 10.1110/ps.9.12.2518.
Internet Resources
  http://sbkb.org/page/show/membprothub
  This site provides data on membrane proteins, 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/
  Detergent suppliers (incomplete list), including detailed information on detergent properties (sometimes also downloadable brochures)
  http://www.merckmillipore.com/
  https://www.sigmaaldrich.com/
  http://www.bachem.com/
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library