Isolation of Organelles and Prefractionation of Protein Extracts Using Free‐Flow Electrophoresis

Peter J. A. Weber1, Gerhard Weber1, Christoph Eckerskorn1

1 Tecan Munich GmbH, Kirchheim
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 22.5
DOI:  10.1002/0471140864.ps2205s32
Online Posting Date:  August, 2004
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Abstract

One of the major obstacles in the analysis of proteomes is the extreme complexity of any particular cell or biological fluid. Free‐flow electrophoresis (FFE) is a powerful tool for reduction of this complexity, which is a prerequisite for systematic and comprehensive protein analyses. Protocols are provided in this unit for sample fractionation at two different stages: on the protein level by isoelectric focusing FFE fractionation of crude protein mixtures such as whole cell lysates, and on a subcellular level by zone‐electrophoretic FFE purification of organelles.

Keywords: Free‐flow electrophoresis; fractionation; organelles; protein; proteomics; isoelectric focusing; zone electrophoresis

     
 
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Table of Contents

  • Basic Protocol 1: Native FFE Fractionation of Crude Protein Mixtures Using the Isoelectric Focusing Mode
  • Alternate Protocol 1: Denaturing FFE Fractionation of Crude Protein Mixtures Using the Isoelectric Focusing Mode
  • Basic Protocol 2: FFE Purification of Organelles Using the Zone Electrophoresis Mode
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Native FFE Fractionation of Crude Protein Mixtures Using the Isoelectric Focusing Mode

  Materials
  • 1:1 (w/w) glycerol/isopropanol
  • Isopropanol (2‐propanol)
  • Kerosene, low‐odor
  • ProTeam FFE SPADNS (sulfanilic acid azochromotrop; Tecan)
  • Anode circuit solution (100 mM H 2SO 4; see recipe)
  • Cathode circuit solution (100 mM NaOH; see recipe)
  • Anode stabilization medium for protocol 1 (see recipe)
  • Separation medium 1 for protocol 1 (see recipe)
  • Separation medium 2 for protocol 1 (see recipe)
  • Separation medium 3 for protocol 1 (see recipe)
  • Cathode stabilization medium for protocol 1 (see recipe)
  • Counterflow medium for protocol 1 (see recipe)
  • ProTeam FFE pI marker (Tecan)
  • Concentrated protein solution for analysis
  • ProTeam FFE system (Tecan) equipped with:
    • Recirculating water cooler and 6‐mm i.d. tubing (supplied with instrument, but other water cooler of power ≥350 W may be used)
    • 7 × 0.64–mm i.d. (orange‐white) media tubes
    • 1 × 1.42–mm i.d. (yellow‐yellow) counterflow tube
    • 1 × 0.51–mm i.d. (orange‐yellow) sample tube
    • 0.4‐mm spacer
    • Anode and cathode membranes
    • 0.65‐mm filter paper strips
  • Lint‐free paper towels
  • Balance capable of weighing ∼10 mg to 2 kg
  • 96‐well microtiter plates
  • 96‐well deep‐well plates
  • 96‐well absorbance reader (optional; for quality controls) equipped with two filters (λ abs= 500 to 530 nm and λ abs= 400 to 420 nm)

Alternate Protocol 1: Denaturing FFE Fractionation of Crude Protein Mixtures Using the Isoelectric Focusing Mode

  • Anode stabilization medium for protocol 2 (see recipe)
  • Separation medium 1 for protocol 2 (see recipe)
  • Separation medium 2 for protocol 2 (see recipe)
  • Separation medium 3 for protocol 2 (see recipe)
  • Cathode stabilization medium for protocol 2 (see recipe)
  • Counterflow medium for protocol 2 (see recipe)

Basic Protocol 2: FFE Purification of Organelles Using the Zone Electrophoresis Mode

  Materials
  • 1:1 (w/w) glycerol/isopropanol
  • Isopropanol (2‐propanol)
  • Kerosene low odor
  • ProTeam FFE SPADNS (sulfanilic acid azochromotrop; Tecan)
  • Anode/cathode circuit solution for protocol 3 (see recipe)
  • Anode/cathode stabilization medium for protocol 3 (see recipe)
  • Separation medium for protocol 3 (see recipe)
  • Anode/cathode stabilization medium for protocol 3 (see recipe)
  • Counterflow medium for protocol 3 (see recipe)
  • Organelle suspension for analysis
  • ProTeam FFE system (Tecan) equipped with:
    • Recirculating water cooler and 6‐mm i.d. tubing (supplied with instrument, but other water cooler of power ≥350 W may be used)
    • 7 × 0.64–mm i.d. (orange‐white) media tubes
    • 1 ×1.42–mm i.d. (yellow‐yellow) counterflow tube
    • 1 ×0.25–mm i.d. (orange‐blue) sample tube
    • 0.5‐mm spacer
    • Anode and cathode membranes
    • 0.8‐mm filter paper strips
  • Lint‐free paper towels
  • Balance capable of weighing ∼10 mg to 2 kg within 1 mg of accuracy
  • 96‐well microtiter plates
  • 96‐well deep‐well plates
  • 96‐well absorbance reader (optional) equ ipped with an appropriate filter for light scattering experiments (λ abs ∼420 nm)
  • Additional reagents and equipment for purification of organelles (unit 4.2)
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Figures

Videos

Literature Cited

   Amigorena, S., Drake, J.R., Webster, P., and Mellman, I. 1994. Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes. Nature 369:113‐119.
   Baldermann, C., Lupas, A., Lubieniecki, J., and Engelhardt, H. 1998. The regulated outer membrane protein Omp21 from Comamonas acidovorans is identified as a member of a new family of eight‐stranded beta‐sheet proteins by its sequence properties. J. Bacteriol. 180:3741‐3749.
   Bard, F., Patel, U., Levy, J.B., Horne, W.C., and Baron, R. 2002. Molecular complexes that contain both c‐Cbl and c‐Src associate with Golgi membranes. Eur. J. Cell. Biol. 81:26‐35.
   Barrollier, J., Watzke, E., and Gibian, H. 1958. Einfache Apparatur für die trägerfreie präparative Durchlauf‐Elektrophorese. Z. Naturforschung 13b:754‐755.
   Bernardo, K., Krut, O., Wiegmann, K., Kreder, D., Micheli, M., Schafer, R., Sickman, A., Schmidt, W.E., Schroder, J.M., Meyer, H.E., Sandhoff, K., and Kroenke, M. 2000. Purification and characterization of a magnesium‐dependent neutral sphingomyelinase from bovine brain. J. Biol. Chem. 275:7641‐7647.
   Hannig, K. 1961. Die trägerfreie kontinuierliche Elektrophorese und ihre Anwendung. Z. Anal. Chem. 181:244‐254.
   Hoffmann, P., Ji, H., Moritz, R.L., Connolly, L.M., Frecklington, D.F., Layton, M.J., Eddes, J.S., and Simpson, R.J. 2001. Continuous free‐flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: A non‐two‐dimensional gel electrophoresis‐based proteome analysis strategy. Proteomics 1:807‐818.
   Kushimoto, T., Basrur, V., Valencia, J., Matsunaga, J., Vieira, W.D., Ferrans, V.J., Muller, J., Appella, E., and Hearing, V.J. 2001. A model for melanosome biogenesis based on the purification and analysis of early melanosomes. Proc. Natl. Acad. Sci. U.S.A. 98:10698‐10703.
   Lasch, J., Moschner, S., Sann, H., Zellmer, S., and Koelsch, R. 1998. Aminopeptidase P−A cell surface antigen of endothelial and lymphoid cells: Catalytical and immuno‐histotopical evidences. Biol. Chem. 379:705‐709.
   Maida, R., Krieger, J., Gebauer, T., Lange, U., and Ziegelberger, G. 2000. Three pheromone‐binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi. Eur. J. Biochem. 267:2899‐2908.
   Mohr, H., and Voelkl, A. 2002. Isolation of peroxisomal subpopulations from mouse liver by immune free‐flow electrophoresis. Electrophoresis 23:2130‐2137.
   Pierre, P., Turley, S.J., Gatti, E., Hull, M., Meltzer, J., Mirza, A., Inaba, K., Steinman, R.M., and Mellman, I. 1997. Developmental regulation of MHC class II transport in mouse dendritic cells. Nature 388:787‐792.
   Song, J.‐F., Liu, T., Shen, X., Wu, G.‐D., and Xia, Q.‐C. 1998. Application of free‐flow electrophoresis to the purification of trichosanthin from a crude product of acetone fractional precipitation. Electrophoresis 19:1097‐1103.
   Thery, C., Boussac, M., Veron, P., Ricciardi‐Castagnoli, P., Raposo, G., Garin, J., and Amigorena, S. 2001. Proteomic analysis of dendritic cell‐derived exosomes: A secreted subcellular compartment distinct from apoptotic vesicles. J. Immunol. 166:7309‐7318.
Key References
   Krivankova, L. and Bocek, P. 1998. Continuous free‐flow electrophoresis. Electrophoresis 19:1064‐1074.
  Excellent review of the principles of the method, the possible techniques and a variety of applications.
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