Optimization of 2-dimensional gel electrophoresis for proteomic studies of solid tumor tissue samples

Liang,Xu; Wang,Jing-Rong; Wong,Kam-Wai  V.; Hsiao,Wen  Luan; Zhou,Hua; Jiang,Zhi-Hong; Kam,Kin   Ting R.; Liu,Liang

doi:10.3892/mmr.2013.1815

2014-February Volume 9 Issue 2

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2014-February Volume 9 Issue 2

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Article

Optimization of 2-dimensional gel electrophoresis for proteomic studies of solid tumor tissue samples

Authors:
- Xu Liang
- Jing-Rong Wang
- Kam-Wai V. Wong
- Wen Luan Hsiao
- Hua Zhou
- Zhi-Hong Jiang
- Kin Ting R. Kam
- Liang Liu
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Affiliations: State Key Laboratory of Quality Research in Chinese Medicine/Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau 999078, Macau SAR, P.R. China, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong 999077, SAR, P.R. China
Pages: 626-632
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Published online on: November 20, 2013

https://doi.org/10.3892/mmr.2013.1815
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Abstract

The method of 2‑dimensional gel electrophoresis (2-DE) has been widely used for the proteomic profiling of solid biological samples, however, the analytical conditions have not been optimized. The present study optimized the major conditions of 2‑DE for determining the protein contents of solid tumor tissues, through enhancement of the separation efficiency and resolution. Three major analytical conditions of 2‑DE analysis, namely protein extraction, focusing time for isoelectric focusing (IEF), and pre‑reduction and alkylation prior to IEF, were carefully examined so that the optimal parameters and procedures were achieved. The use of a bead mill for protein extraction resulted in a higher protein yield in a minimal processing time. An optimal focusing time for IEF was established which improved the 2‑DE image quality and reproducibility. Furthermore, reduction and alkylation of the protein sample prior to IEF reduced the horizontal streaking caused by oxidation and improved the resolution at the cathode. The optimized 2‑DE analysis enabled the detection of 20% more protein spots compared with the previous reported conditions, with higher image quality and reproducibility. Accordingly, the optimized conditions may be used in the 2‑DE analysis of tumor tissue samples, by which novel biomarkers of cancerous diseases and molecular targets of drugs are expected to be identified.

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1	Klose J: Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik. 26:231–243. 1975.
2	O’Farrell PH: High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 250:4007–4021. 1975.
3	Görg A, Obermaier C, Boguth G, et al: The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 21:1037–1053. 2000.
4	Campostrini N, Areces LB, Rappsilber J, et al: Spot overlapping in two-dimensional maps: a serious problem ignored for much too long. Proteomics. 5:2385–2395. 2005. View Article : Google Scholar : PubMed/NCBI
5	Galvani M, Hamdan M and Righetti PG: Investigating the reaction of a number of gel electrophoresis cross-linkers with beta-lactoglobulin by matrix assisted laser desorption/ionization-mass spectrometry. Electrophoresis. 21:3684–3692. 2000. View Article : Google Scholar
6	Shevchenko A, Tomas H, Havlis J, Olsen JV and Mann M: In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 1:2856–2860. 2006. View Article : Google Scholar : PubMed/NCBI
7	Herbert B, Righetti P, Citterio A and Boschetti E: Sample preparation and prefractionation techniques for electrophoresis-based proteomics. Proteome Research: Concepts, Technology and Application. Wilkins MR, Appel RD, Williams KL and Hochstrasser DF: 2nd edition. Springer; Heidelberg Berlin: pp. 15–40. 2007, View Article : Google Scholar
8	Rabilloud T: Solubilization of proteins in 2-D electrophoresis. An outline. Methods Mol Biol. 112:9–19. 1999.PubMed/NCBI
9	Görg A, Postel W and Günther S: The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 9:531–546. 1988.
10	Görg A: Two-dimensional electrophoresis. Nature. 349:545–546. 1991.
11	Görg A, Boguth G, Obermaier C, Posch A and Weiss W: Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): the state of the art and the controversy of vertical versus horizontal systems. Electrophoresis. 16:1079–1086. 1995.PubMed/NCBI
12	Herbert B, Galvani M, Hamdan M, et al: Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how? Electrophoresis. 22:2046–2057. 2001. View Article : Google Scholar : PubMed/NCBI
13	Candiano G, Bruschi M, Musante L, et al: Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis. 25:1327–1333. 2004. View Article : Google Scholar : PubMed/NCBI

Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Optimization of 2-dimensional gel electrophoresis for proteomic studies of solid tumor tissue samples

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