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Two-Dimensional Gel Electrophoresis-Based Proteomic Analysis of Brain Synapses

  • Karl-Heinz SmallaEmail author
  • Ursula Wyneken
Protocol
Part of the Neuromethods book series (NM, volume 57)

Abstract

Neuroproteomic technologies are crucial for a deeper understanding of molecular mechanisms underlying neuronal plasticity and pathologies of the central nervous system. For a comprehensive neuroproteomic analysis, high-resolution high-throughput techniques are indispensable. The most commonly used ­system for 2D gel electrophoresis is based on the combination of isoelectric focussing and sodium dodecyl ­sulphate-polyacrylamide gel electrophoresis. Nevertheless, the analysis of complex samples derived from biochemically prepared synaptic fractions raises some crucial challenges. Therefore, we describe the ­general 2D gel electrophoresis procedure, including sample preparation, gel casting, electrophoresis ­condition, and the detection of proteins on the gel with mass spectrometry compatible silver staining, sypro-ruby or colloidal Coomassie Brilliant Blue.

Key words

2D gel electrophoresis Sample preparation Isoelectric focussing SDS-PAGE Protein staining Image analysis 

Notes

Acknowledgements

This work was supported by bilateral programs of the Deutsche Forschungsgemeinschaft (DFG 444 CHL-113/32/0-1) and the Bundesministerium für Bildung und Forschung (BMBF CHL 06/027) with Conicyt (Chile), Proyecto Anillo 09-06 (PBCT, Conicyt, Chile) and the European Union (ZVOH-TP3/2, ZVOH 6/1).

References

  1. 1.
    Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26:231–243.Google Scholar
  2. 2.
    O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021.PubMedGoogle Scholar
  3. 3.
    Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231.PubMedCrossRefGoogle Scholar
  4. 4.
    Wittig I, Braun HP, Schägger H (2006) Blue native PAGE. Nat Protoc 1:418–428.PubMedCrossRefGoogle Scholar
  5. 5.
    Hartinger J, Stenius K, Hogemann 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
  6. 6.
    Burre J, Volknandt W (2007) The synaptic vesicle proteome. J Neurochem 101:1448–1462.PubMedCrossRefGoogle Scholar
  7. 7.
    Wenge B, Bonisch H, Grabitzki J, Lochnit G, Schmitz B, Ahrend MH (2008) Separation of membrane proteins by two-dimensional electrophoresis using cationic rehydrated strips. Electrophoresis 29:1511–1517.PubMedCrossRefGoogle Scholar
  8. 8.
    Suzuki T (2011) Isolation of synapse sub-domains by subcellular fractionation using sucrose density gradient centrifugation. Chapter 4 of this book.Google Scholar
  9. 9.
    Carlin RK, Grab DJ, Cohen RS, Siekevitz P (1980) Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities. J Cell Biol 86:831–845.PubMedCrossRefGoogle Scholar
  10. 10.
    Smalla KH, Matthies H, Langnase K, Shabir S, Bockers TM, Wyneken U, Staak S, Krug M, Beesley PW, Gundelfinger ED (2000) The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses. Proc Natl Acad Sci USA 97:4327–4332.PubMedCrossRefGoogle Scholar
  11. 11.
    Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858.PubMedCrossRefGoogle Scholar
  12. 12.
  13. 13.
    Heukeshoven J, Dernick R (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6:103–112.CrossRefGoogle Scholar
  14. 14.
    Swatton JE, Prabakaran S, Karp NA, Lilley KS, Bahn S (2004) Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Mol Psychiatry 9:128–143.PubMedCrossRefGoogle Scholar
  15. 15.
    GE Healthcare Web page http://www1gelifesciencescom/aptrix/upp00919nsf/Content/Proteomics%20DIGE%7EProteomics%20DIGE%20Introduction?OpenDocument&hometitle=Proteomics or DyeAGNOSTICS Web page: http://www.dyeagnostics.com/wp/?page_id=6.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Leibniz Institute for NeurobiologyMagdeburgGermany

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