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

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Book cover Difference Gel Electrophoresis (DIGE)

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

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.

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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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Acknowledgments

This work was supported, in part, by the National Health & Medical Research Council of Australia (program grant #487922 (R.J.S)), and funds from the Operational Infrastructure Support Program provided by the Victorian Government of Australia.

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Authors and Affiliations

  1. Ludwig Institute for Cancer Research, Parkville, VIC, Australia

    Rommel A. Mathias, Hong Ji & Richard J. Simpson

Authors
  1. Rommel A. Mathias
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  2. Hong Ji
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  3. Richard J. Simpson
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Corresponding author

Correspondence to Richard J. Simpson .

Editor information

Editors and Affiliations

  1. BioCentre, University of Reading, Reading, United Kingdom

    Rainer Cramer

  2. SERVA Electrophoresis GmbH, Carl-Benz-Strasse 7, Heidelberg, 69115, Germany

    Reiner Westermeier

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Mathias, R.A., Ji, H., Simpson, R.J. (2012). Proteomic Profiling of the Epithelial-Mesenchymal Transition Using 2D DIGE. In: Cramer, R., Westermeier, R. (eds) Difference Gel Electrophoresis (DIGE). Methods in Molecular Biology, vol 854. Humana Press. https://doi.org/10.1007/978-1-61779-573-2_19

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  • DOI: https://doi.org/10.1007/978-1-61779-573-2_19

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-572-5

  • Online ISBN: 978-1-61779-573-2

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