Two-Dimensional BAC-SDS Polyacrylamide Gel Electrophoresis for the Fractionation and Identification of Synaptic Vesicle Proteins and the Presynaptic Active Zone

  • Joern Barth
  • Walter VolknandtEmail author
Part of the Neuromethods book series (NM, volume 57)


Recent progress in mass spectrometry allowed the identification of numerous proteins in complex soluble mixtures; however, the analysis of membranous subproteomes is still a challenging task. Integral membrane proteins, in particular those with more than one transmembrane domain, have been difficult to separate by 2D electrophoretic methods due to their tendency to aggregate. The conventionally used 2D technique based on isoelectric focussing in the first-dimension and SDS-PAGE in the second-dimension is a powerful method to separate soluble protein samples, albeit it has major drawbacks in the separation of hydrophobic proteins. An alternative electrophoretic technique especially suitable for the analytical and preparative separation of membrane proteins is 2D 16-BAC/SDS electrophoresis. The first-­dimension involves discontinuous gel electrophoresis towards the cathode in an acidic buffer system using the ­cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC). The separation in the second-dimension towards the anode is based on the anionic detergent SDS. Using this system in combination with subsequent MALDI-TOF mass spectrometry enabled us to identify the proteinaceous inventory of synaptic vesicles from rat brain. Moreover, applying these techniques to plasma membrane-docked synaptic vesicles allowed us to identify numerous proteins of the presynaptic active zone. Here, we describe the application of 2D 16-BAC-SDS polyacrylamide gel electrophoresis for the fractionation and identification of synaptic vesicle proteins and the presynaptic active zone.

Key words

16-BAC Protein separation 2D gel electrophoresis Synaptic vesicles proteins Active zone Cationic detergents SDS-PAGE 



We are indebted to Drs Jaqueline Burré and Marco Morciano for sharing data with us.


  1. 1.
    Littleton, J. T. (2006) Mixing and matching during synaptic vesicle endocytosis. Neuron 51, 149–151.PubMedCrossRefGoogle Scholar
  2. 2.
    Ryan, T. A. (2006) A pre-synaptic to-do list for coupling exocytosis to endocytosis. Curr. Opin. Cell Biol. 18, 416–421.PubMedCrossRefGoogle Scholar
  3. 3.
    Li, L., Chin, L.-S. (2003) The molecular machinery of synaptic vesicle exocytosis. Cell. Mol. Life Sci. 60, 942-960.PubMedGoogle Scholar
  4. 4.
    Südhof, T. C. (2004) The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509–547.PubMedCrossRefGoogle Scholar
  5. 5.
    O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.Google Scholar
  6. 6.
    Rabilloud, T., Blisnick, T., Heller, M., Luche, S., Aebersold, R., Lunardi, J., Braun-Breton C. (1999) Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts. Electrophoresis 20, 3603–3610.PubMedCrossRefGoogle Scholar
  7. 7.
    Santoni, V., Molloy, M., Rabilloud T. (2000) Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054–1070.PubMedCrossRefGoogle Scholar
  8. 8.
    Kashino, Y. (2003) Separation methods in the analysis of protein membrane complexes. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 797, 191–216.PubMedCrossRefGoogle Scholar
  9. 9.
    Luche, S., Santoni, V., Rabilloud T. (2003) Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in twodimensional electrophoresis. Proteomics 3, 249–253.PubMedCrossRefGoogle Scholar
  10. 10.
    Burré, J., Volknandt, W. (2007) The ­synaptic vesicle proteome. J. Neurochem. 101,1448–1462.PubMedCrossRefGoogle Scholar
  11. 11.
    Braun, R. J., Kinkl, N., Beer, M., Ueffing, M. (2007) Two-dimensional electrophoresis of membrane proteins. Anal. Bioanal. Chem. 389, 1033–1045.PubMedCrossRefGoogle Scholar
  12. 12.
    Macfarlane, D. E. (1983) Use of benzyldimethyl-n-hexadecylammonium chloride (“16-BAC”), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells. Anal. Biochem. 132, 231–235.PubMedCrossRefGoogle Scholar
  13. 13.
    Macfarlane, D. E. (1989) Two dimensional benzyldimethyl-n-hexadecylammonium ­chloride → sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis: a high capacity high resolution technique for the purification of proteins from complex mixtures. Anal. Biochem. 176, 457–463.PubMedCrossRefGoogle Scholar
  14. 14.
    Hartinger, J., Stenius, K., Högemann, D., Jahn, R. (1996) 16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins. Anal. Biochem. 240, 126–133.PubMedCrossRefGoogle Scholar
  15. 15.
    Peters, C., Bayer, M. J., Bühler, S., Andersen, J. S., Mann, M., Mayer, A. (2001) Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature 409, 581–588.PubMedCrossRefGoogle Scholar
  16. 16.
    Yamaguchi, Y., Miyagi, Y., Baba, H. (2008) Two-dimensional electrophoresis with cationic detergents, a powerful tool for the proteomic analysis of myelin proteins. Part 1: technical aspects of electrophoresis. J. Neurosci. Res. 86, 755–765.PubMedCrossRefGoogle Scholar
  17. 17.
    Zahedi, R. P., Meisinger, C., Sickmann, A. (2005) Two-dimensional benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE for membrane proteomics. Proteomics 5, 3581–3588.PubMedCrossRefGoogle Scholar
  18. 18.
    Morciano, M., Burré, J., Corvey, C., Karas, M., Zimmermann, H., Volknandt, W. (2005) Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis. J. Neurochem. 95, 1732–1745.PubMedCrossRefGoogle Scholar
  19. 19.
    Morciano, M., Beckhaus, T., Karas, M., Zimmermann, H., Volknandt, W. (2009) The proteome of the presynaptic active zone: from docked synaptic vesicles to adhesion molecules and maxi-channels. J. Neurochem. 108, 662–675.PubMedCrossRefGoogle Scholar
  20. 20.
    Burré, J., Beckhaus, T., Schägger, H., Corvey, C., Hofmann, S., Karas, M., Zimmermann, H., Volknandt, W. (2006) Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques. Proteomics 6, 6250–6262.PubMedCrossRefGoogle Scholar
  21. 21.
    Rais, I., Karas, M., Schägger, H. (2004). Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification. Proteomics 4, 2567–2571.PubMedCrossRefGoogle Scholar
  22. 22.
    Wessel, D., Flügge, U. I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and ­lipids. Anal. Biochem. 138, 141–143.PubMedCrossRefGoogle Scholar
  23. 23.
    Eley, M. H., Burns, P. C., Kannapell, C. C., Champbell, P. S. (1979) Cetyltrimethylammo­nium bromide polyacrylamide gel electrophoresis: estimation of protein subunit molecular weights using cationic detergents. Anal. Biochem. 181, 411–419.CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.AK NeurochemistryBiocenter of JW Goethe-UniversityFrankfurt/MainGermany

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