Difference Gel Electrophoresis (DIGE) pp 269-286

Part of the Methods in Molecular Biology book series (MIMB, volume 854)

Proteomic Profiling of the Epithelial-Mesenchymal Transition Using 2D DIGE

Protocol

Abstract

Metastasis remains the primary cause of cancer patient death. Although the precise molecular mechanisms at play remain largely unknown, tumor progression is currently hypothesized to follow a series of sequential steps known as the metastatic cascade. An important component, thought to be involved early in this cascade, is the process known as epithelial-mesenchymal transition (EMT), whereby epithelial cells undergo morphogenetic alterations and acquire properties typical of mesenchymal cells. EMT confers a metastatic advantage to the cancer cells through the loss of cell-cell adhesion, enhanced proteolytic activity, and increased cell migration and invasiveness. This chapter describes the experimental workflow for the secretome analysis of MDCK cells undergoing oncogenic Ras, and Ras/TGF-β-mediated EMT. To enable this comparison, serum-free cell culture conditions were optimized, and a secretome purification methodology established. Secretome samples were then subjected to DIGE analysis to reveal and quantify proteins that are differentially expressed during EMT. The proteomic strategy detailed within successfully identified several EMT modulators and broadens our understanding of the extracellular facets of the EMT process.

Key words

Epithelial-mesenchymal transition EMT MDCK DIGE Ras Secretome Quantitative proteomics 

Abbreviations

2DE

Two-dimensional gel electrophoresis

APS

Ammonium persulfate

BVA

Biological variation analysis

CM

Conditioned medium

DIA

Differential in-gel analysis

DIGE

Two-dimensional fluorescence difference gel electrophoresis

DMEM

Dulbecco’s Modified Eagle Medium

DMF

Dimethylformamide

DTT

Dithiothreitol

EDA

Extended data analysis

EMT

Epithelial-mesenchymal transition

FCS

Fetal calf serum

LC

Liquid chromatography

MDCK

Madin Darby canine kidney

MS

Mass spectrometry

RT

Room temperature

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Rommel A. Mathias
    • 1
  • Hong Ji
    • 1
  • Richard J. Simpson
    • 1
  1. 1.Ludwig Institute for Cancer ResearchParkvilleAustralia

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