Abstract

Quantitative 2D-gel-dependent proteomics became feasible with 2D fluorescence difference gel electrophoresis (2D-DIGE), and this technique has gained wide acceptance because it has eliminated the gel to gel variations and greatly facilitated the quantitative comparisons across gels for many different experimental conditions. However, the co-migration of several proteins in the same spot is still a major limitation which detracts from the accuracy of comparative quantification and prevents unambiguous post- translational modifications (PTMs) detection. A protocol based on traditional polyacrylamide gel IEF sample fractionation, and followed by two consecutive SDS-PAGE electrophoreses alleviates co-migration limitations. The use of two different buffer systems for SDS-PAGE is central to the proposed approach.

Original languageEnglish
Pages (from-to)427-440
Number of pages14
JournalMethods in Molecular Biology
Volume1295
DOIs
Publication statusPublished - 4 Dec 2014

Fingerprint

Proteome
Electrophoresis
Gels
Polyacrylamide Gel Electrophoresis
Post Translational Protein Processing
Proteomics
Buffers
Fluorescence
Proteins

Keywords

  • 2D-DIGE
  • 3D-DIGE
  • Co-migration
  • Complex proteomes
  • Western blot

Cite this

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title = "Three-dimensional electrophoresis for quantitative profiling of complex proteomes",
abstract = "Quantitative 2D-gel-dependent proteomics became feasible with 2D fluorescence difference gel electrophoresis (2D-DIGE), and this technique has gained wide acceptance because it has eliminated the gel to gel variations and greatly facilitated the quantitative comparisons across gels for many different experimental conditions. However, the co-migration of several proteins in the same spot is still a major limitation which detracts from the accuracy of comparative quantification and prevents unambiguous post- translational modifications (PTMs) detection. A protocol based on traditional polyacrylamide gel IEF sample fractionation, and followed by two consecutive SDS-PAGE electrophoreses alleviates co-migration limitations. The use of two different buffer systems for SDS-PAGE is central to the proposed approach.",
keywords = "2D-DIGE, 3D-DIGE, Co-migration, Complex proteomes, Western blot",
author = "Sergio Mauro and Bertrand Colignon and Marc Dieu and Edouard Delaive and Martine Raes",
year = "2014",
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AU - Mauro, Sergio

AU - Colignon, Bertrand

AU - Dieu, Marc

AU - Delaive, Edouard

AU - Raes, Martine

PY - 2014/12/4

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AB - Quantitative 2D-gel-dependent proteomics became feasible with 2D fluorescence difference gel electrophoresis (2D-DIGE), and this technique has gained wide acceptance because it has eliminated the gel to gel variations and greatly facilitated the quantitative comparisons across gels for many different experimental conditions. However, the co-migration of several proteins in the same spot is still a major limitation which detracts from the accuracy of comparative quantification and prevents unambiguous post- translational modifications (PTMs) detection. A protocol based on traditional polyacrylamide gel IEF sample fractionation, and followed by two consecutive SDS-PAGE electrophoreses alleviates co-migration limitations. The use of two different buffer systems for SDS-PAGE is central to the proposed approach.

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