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2-D Gel Electrophoresis: Constructing 2D-Gel Proteome Reference Maps

  • Maria Paola Simula
  • Agata Notarpietro
  • Giuseppe Toffoli
  • Valli De Re
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 815)

Abstract

Two-dimensional gel electrophoresis (2-DE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomic technologies. Based on two independent biochemical characteristics of proteins, it combines isoelectric focusing, which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. An evolution of conventional 2-DE is represented by the 2D-Difference in Gel Electrophoresis (2D-DIGE) that allows sample multiplexing and achieving more accurate and sensitive quantitative proteomic determinations. The 2-DE separation permits the generation of protein maps of different cells or tissues and the study, by differential proteomics, of protein expression changes associated to the different states of a biological system. In order to identify the molecular bases of pathological processes, it is also useful to characterize the physiological protein homeostasis in healthy cells or tissues. On these grounds, the availability of detailed 2D reference maps could be very useful for proteomic studies. The protocol described in this chapter is based on the 2D-DIGE technology and has been applied to obtain the first 2-DE reference map of the human small intestine.

Key words

Reference map 2D-DIGE 2D-electrophoresis MALDI-TOF Proteome 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Maria Paola Simula
    • 1
  • Agata Notarpietro
    • 1
  • Giuseppe Toffoli
    • 1
  • Valli De Re
    • 1
  1. 1.Experimental and Clinical Pharmacology Unit, CRO Centro di Riferimento Oncologico, IRCCS National Cancer InstituteAviano (PN)Italy

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