Preparing Protein Extracts for Quantitative Two‐Dimensional Gel Comparison

Cécile Lelong1, Thierry Rabilloud1

1 University Grenoble Alpes, CNRS and CEA UMR 5249, Laboratory of Chemistry and Biology of Metals, iRTSV/LCBM, CEA Grenoble
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 22.4
DOI:  10.1002/0471140864.ps2204s80
Online Posting Date:  April, 2015
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Abstract

This unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids. The optimized extraction process is strongly sample dependent and cannot be described for every type of sample. Instead, typical protocols are provided as general guidelines and illustrate good starting points for optimization of sample preparation. These solubilization procedures take into account the constraints imposed by two‐dimensional electrophoresis and are thus well suited for proteomic approaches. © 2015 by John Wiley & Sons, Inc.

Keywords: proteomics; sample preparation; two‐dimensional electrophoresis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Protein Extraction from Cultured Animal Cells, Cell Nuclei, and Bacteria
  • Alternate Protocol 1: Protein Extraction from Animal Tissues
  • Alternate Protocol 2: Protein Extraction from Plasma or Serum
  • Alternate Protocol 3: Protein Extraction from Other Biological Fluids by Precipitation
  • Basic Protocol 2: Protein Extraction from Plant Tissues and Cells
  • Alternate Protocol 4: Protein Extraction from Plant Cells and Tissues by Phenol‐Methanol
  • Basic Protocol 3: Protein Extraction from Subcellular Organelles Other than Nuclei
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Protein Extraction from Cultured Animal Cells, Cell Nuclei, and Bacteria

  Materials
  • Animal or bacterial cells, or cell nuclei (unit 4.2; Castle, )
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Isotonic buffer (see recipe)
  • 1.25 × lysis buffer (see recipe)
  • 40% carrier ampholytes (GE Healthcare), pI range 3 to 10
  • Thick‐walled polyallomer ultracentrifuge tube
  • Thick laboratory plastic film
  • Ultracentrifuge with fixed‐angle rotor
  • Additional reagents and equipment for fractionation of cell nuclei (unit 4.2; Castle, ) and measuring protein concentration (unit 3.4; Olson and Markwell, )

Alternate Protocol 1: Protein Extraction from Animal Tissues

  Additional Materials (also see protocol 1)
  • Animal tissue, frozen
  • Tissue homogenizer adapted to the hardness of the tissue

Alternate Protocol 2: Protein Extraction from Plasma or Serum

  Additional Materials (also see protocol 1)
  • Serum or plasma
  • SDS buffer (see recipe)
  • 1.25 × denaturing buffer (see recipe)
  • 20° and 100°C (i.e., boiling) water bath

Alternate Protocol 3: Protein Extraction from Other Biological Fluids by Precipitation

  Additional Materials (also see protocol 1)
  • Biological fluid (other than plasma or serum)
  • 10% (w/v) sodium lauroylsarcosinate solution (see recipe)
  • 100% (6.1 M) trichloroacetic acid
  • Tetrahydrofuran, cold
  • 1× denaturing buffer (see recipe)
  • Refrigerated centrifuge
  • Bath sonicator (optional)

Basic Protocol 2: Protein Extraction from Plant Tissues and Cells

  Materials
  • Plant tissue or cell pellet
  • Liquid N 2
  • Acid denaturing solution (see recipe)
  • 0.05% (w/v) DTT in acetone
  • 1 × denaturing buffer (see recipe)
  • Mortar and pestle
  • Ultracentrifuge with fixed‐angle rotor
  • Additional reagents and equipment for measuring protein concentration (unit 3.4; Olson and Markwell, )

Alternate Protocol 4: Protein Extraction from Plant Cells and Tissues by Phenol‐Methanol

  Materials
  • Plant tissue or cell pellet
  • Liquid nitrogen
  • Hypertonic extraction buffer (see recipe)
  • Buffer‐saturated phenol, neutral pH (can be purchased from various suppliers; use phenol saturated with a neutral pH buffer, not an acidic pH buffer)
  • Methanol precipitation solution (see recipe), cold
  • 1 × denaturing buffer (see recipe)
  • Rotary shaker
  • Refrigerated centrifuge
  • Vacuum concentrator
  • Additional reagents and equipment for measuring protein concentration (unit 3.4; Olson and Markwell, )

Basic Protocol 3: Protein Extraction from Subcellular Organelles Other than Nuclei

  Materials
  • Isolated organelles (e.g., unit 4.2)
  • Isotonic buffer (see recipe)
  • 20% (w/v) detergent solution in 50% (v/v) ethanol, 30°C
  • 1.25× chaotrope solution, 30°C (see recipe)
  • Ultracentrifuge with fixed‐angle rotor
  • Additional reagents and equipment for determining protein concentration (unit 3.4; Olson and Markwell, )
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Figures

Videos

Literature Cited

Literature Cited
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  Chevallet M., Diemer, H., Van Dorssealer, A., Villiers, C., and Rabilloud T. 2007. Toward a better analysis of secreted proteins: The example of the myeloid cell secretome. Proteomics 7:1757‐1770.
  Chevallet, M., Santoni, V., Poinas, A., Rouquié, 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.
  Colas des Francs, C., Thiellement, H., and De Vienne, D. 1985. Analysis of leaf proteins by two‐dimensional electrophoresis. Plant Physiol. 78:178‐182.
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  Görg, A., Boguth, G., Obermaier, C., and Weiss, W. 1998. Two‐dimensional electrophoresis of proteins in an immobilized pH 4‐12 gradient. Electrophoresis 19:1516‐1519.
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  Harper, S., Mozdzanowski, J., and Speicher, D. 1998. Two‐dimensional gel electrophoresis. Curr. Protoc. Protein Sci. 11:10.4.1‐10.4.36.
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  Hurkman, W.J. and Tanaka, C.K. 1986. Solubilisation of plant membrane proteins for analysis by two‐dimensional gel electrophoresis. Plant Physiol. 81:802‐806.
  Lelong, C., Chevallet, M., Diemer, H., Luche, S., Van Dorsselaer, A., and Rabilloud, T. 2012. Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloid cell lines nuclear proteomes. J. Proteomics 77:577‐602.
  Luche, S., Santoni, V., and Rabilloud, T. 2003. Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two‐dimensional electrophoresis. Proteomics 3:249‐253.
  Molloy, M.P., Herbert, B.R., Williams, K.L., and Gooley, A.A. 1999. Extraction of Escherichia coli proteins with organic solvents prior to two‐dimensional electrophoresis. Electrophoresis 20:701‐704.
  Olson, B.J. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. in Protein Sci. 48:3.4.1‐3.4.29.
  Rabilloud, T., Hubert, M., and Tarroux, P. 1986. Procedures for two‐dimensional electrophoretic analysis of nuclear proteins. J. Chromatogr. 351:77‐89.
  Rabilloud, T., Adessi, C., Giraudel, A., and Lunardi, J. 1997. Improvement of the solubilization of proteins in two‐dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18:307‐316.
  Tastet, C., Charmont, S., Chevallet, M., Luche, S., and Rabiloud, T. 2003. Structure‐efficiency relationships of zwitterionic detergents as protein solubilizers in two‐dimensional electrophoresis. Proteomics 3:111‐121.
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