Advertisement

Application of 2D DIGE in Animal Proteomics

  • Ingrid MillerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 854)

Abstract

Two-dimensional electrophoresis (2 DE) is one of the most important proteomic tools and allows studying the complexity of proteomes of different origin. This chapter describes a setup for 2D DIGE with minimal labeling for qualitative and quantitative applications. It relies on homemade gels of medium size and in our hands has been found useful for a wide variety of separation problems involving complex protein mixtures of animal or human origin. The basic method is given for serum proteins of different species, but with minor modifications the method may be easily adapted to other sample materials (other body fluids, cells, tissues), conditions, or size. Examples are given for simple pattern comparisons (e.g., quality control, fast comparison of just two samples) as well as for quantitative applications to larger sample sets.

Key words

2D DIGE Animal body fluids Animal sera Animal tissues Homemade IPGs 

References

  1. 1.
    Westermeier R, Naven T, Höpker H-R (2008) Proteomics in Practice. A Guide to Successful Experimental Design, 2nd edn. Wiley-VCH Verlagsgesellschaft GmbH, Weinheim, Germany.Google Scholar
  2. 2.
    Uenlue M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077.CrossRefGoogle Scholar
  3. 3.
    Timms JF, Cramer R (2008) Difference gel electrophoresis. Proteomics 8, 4886–4897.PubMedCrossRefGoogle Scholar
  4. 4.
    Miller I, Friedlein A, Tsangaris G, Maris A, Fountoulakis M, Gemeiner M (2004) The serum proteome of Equus caballus. Proteomics 4, 3227–3234.PubMedCrossRefGoogle Scholar
  5. 5.
    Miller I, Teinfalt M, Leschnik M, Wait R, Gemeiner M (2004) Nonreducing two-dimensional gel electrophoresis for the detection of Bence Jones proteins in serum and urine. Proteomics 4, 257–260.PubMedCrossRefGoogle Scholar
  6. 6.
    Kozlov AV, Duvigneau JC, Miller I, Nürnberger S, Gesslbauer B, Kungl A, Öhlinger W, Hartl RT, Gille L, Staniek K, Gregor W, Haindl S, Redl H (2009) Endotoxin causes functional endoplasmic reticulum failure, possibly mediated by mitochondria. Biochim Biophys Acta - Mol Basis Dis 1792, 521–530.Google Scholar
  7. 7.
    Miller I, Crawford J, Gianazza E (2006) Protein stains for proteomic applications: Which, when, why? Proteomics 6, 5385–5408.PubMedCrossRefGoogle Scholar
  8. 8.
    Radwan M, Miller I, Grunert T, Marchetti M, Vogl C, O’Donoghue N, Dunn MJ, Kolbe T, Allmaier G, Gemeiner M, Müller M, Strobl B (2008) The impact of Tyrosine kinase 2 (Tyk2) on the proteome of murine macrophages and their response to lipopolysaccharide (LPS). Proteomics 8, 3469–3485.PubMedCrossRefGoogle Scholar
  9. 9.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.PubMedCrossRefGoogle Scholar
  10. 10.
    Miller I, Eberini I, Gianazza E (2010) Other than IPG-DALT: two-dimensional electrophoresis variants. Proteomics 10, 586–610.PubMedCrossRefGoogle Scholar
  11. 11.
    Gianazza E, Celentano F, Magenes S, Ettori C, Righetti PG (1989) Formulations for immobilized pH gradients including pH extremes. Electrophoresis 10, 806–808.PubMedCrossRefGoogle Scholar
  12. 12.
    Hoving S, Voshol H, van Oostrum J (2000) Towards high performance two-dimensional gel electrophoresis using ultrazoom gels. Electrophoresis 21, 2617–2621.PubMedCrossRefGoogle Scholar
  13. 13.
    Tastet C, Lescuyer P, Diemer H, Luche S, van Dorsselaer A, Rabilloud T (2003) A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins. Electro-phoresis 24, 1787–1794.PubMedCrossRefGoogle Scholar
  14. 14.
    Miller I, Radwan M, Strobl B, Müller M, Gemeiner M (2006) Contribution of cell culture additives to the two-dimensional protein patterns of mouse macrophages. Electrophoresis 27, 1626–1629.PubMedCrossRefGoogle Scholar
  15. 15.
    Radwan M, Stiefvater R, Grunert T, Sharif O, Miller I, Marchetti-Deschmann M, Allmaier G, Gemeiner M, Knapp S, Kovarik P, Müller M, Strobl B (2010) Tyrosine kinase 2 controls IL-1beta production at the translational level. J Immunol 185, 3544–3553.PubMedCrossRefGoogle Scholar
  16. 16.
    Miller I, Gemeiner M, Gesslbauer B, Kungl A, Piskernik C, Haindl S, Nürnberger S, Bahrami S, Redl H, Kozlov AV (2006) Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock. FEBS Lett 580, 1257–1262.PubMedCrossRefGoogle Scholar
  17. 17.
    Westermeier R (2004) Method 10: IEF in immobilized pH gradients. In: Westermeier R (ed) Electrophoresis in Practice. A Guide to Methods and Applications of DNA and Protein Separations, 4th edn. VCH Verlagsgesellschaft GmbH, Weinheim, Germany.Google Scholar
  18. 18.
    Molloy MP, Herbert BR, Walsh BJ, Tyler MI, Traini M, Sanchez J-C, Hochstrasser DF, Williams KL, Gooley AA (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19, 837–844.PubMedCrossRefGoogle Scholar
  19. 19.
    Pasquali C, Fialka I, Huber LA (1997) Preparative gel electrophoresis of membrane proteins. Electrophoresis 18, 2573–2581.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department for Biomedical SciencesUniversity of Veterinary Medicine ViennaViennaAustria

Personalised recommendations