6. Two-Dimensional Maps

  1. Mahmoud Hamdan1 and
  2. Pier Giorgio Righetti2

Published Online: 27 JAN 2005

DOI: 10.1002/0471709158.ch6

Proteomics Today: Protein Assessment and Biomarkers Using Mass Spectrometry, 2D Electrophoresis, and Microarray Technology

Proteomics Today: Protein Assessment and Biomarkers Using Mass Spectrometry, 2D Electrophoresis, and Microarray Technology

How to Cite

Hamdan, M. and Righetti, P. G. (2005) Two-Dimensional Maps, in Proteomics Today: Protein Assessment and Biomarkers Using Mass Spectrometry, 2D Electrophoresis, and Microarray Technology, John Wiley & Sons, Inc., Hoboken, NJ, USA. doi: 10.1002/0471709158.ch6

Author Information

  1. 1

    GlaxoSmithKline Research Centre in Verona, Italy

  2. 2

    University of Verona, Italy

Publication History

  1. Published Online: 27 JAN 2005
  2. Published Print: 11 FEB 2005

ISBN Information

Print ISBN: 9780471648178

Online ISBN: 9780471709152

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Keywords:

  • 2-D PAGE;
  • cell disruption;
  • proteolytic attack;
  • precipitation procedures;
  • solubilization cocktail;
  • sequential sample extraction;
  • artefacts;
  • Fata Morganas';
  • sample-prefractionation;
  • rotationally stabilized focusing apparatus;
  • Rotofor;
  • free-flow isoelectric focusing;
  • free-flow electrophoresis;
  • Gradiflow;
  • multicompartment electrolysers;
  • Immobiling membranes;
  • Off-Gel IEF;
  • sub-cellular organelle purification;
  • multidimensional chromatography;
  • protein chips;
  • microarrays;
  • non-denaturing protein maps;
  • spot matching

Summary

In this third and last chapter of the electrophoretic part of this book, the natural evolution of SDS-PAGE and IEF methodology follows its fatal course, i.e. a orthogonal combination of the two techniques (in general IEF as the first, SDS-PAGE as the second) so as to spread maximally all proteins spots obtained from a tissue in the two-dimensional (2D) electrophoretic space. After a glimpse at the early days and at the evolution of 2-D PAGE, the basic methodology pertaining to 2-D PAGE is amply reviewed, starting from methods of cell disruption, potential proteolytic attack during cell disruption, removal of interfering substances, solubilization cocktails, including the most powerful and up to date concoctions. Various techniques of sample application, as well as true artefacts and Fata Morganas' are discussed.

Due to the incredible heterogeneity of the whole proteome of any possible organism, Pre-fractionation tools in proteome analysis are discussed and evaluated, not only those relying on chromatographic means, but also all the variety of methods relying on different electrophoretic

Techniques, including conventional focusing and IPG techniques. Today, of course, pre-fractionation is also based on sub-cellular organelle purification, a technique invented in the 1960 and now experience a strong revival. The pompously called Multidimensional chromatography (two-dimensional, of course) coupled to mass spectrometry is also well described. The book ends with a glimpse at chips and microarrays and with an evaluation of spot matching in 2D gels via commercial software.