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Evaluation of an effective sample prefractionation method for the proteome analysis of breast cancer tissue using narrow range two-dimensional gel electrophoresis.

Abstract
One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4-6.7.
AuthorsKiBeom Lee
JournalBioscience, biotechnology, and biochemistry (Biosci Biotechnol Biochem) Vol. 72 Issue 6 Pg. 1464-74 (Jun 2008) ISSN: 1347-6947 [Electronic] England
PMID18540105 (Publication Type: Journal Article)
Chemical References
  • Buffers
  • Proteome
  • Water
Topics
  • Breast Neoplasms (metabolism)
  • Buffers
  • Cell Line, Tumor
  • Electrophoresis, Gel, Two-Dimensional (methods)
  • Humans
  • Hydrogen-Ion Concentration
  • Isoelectric Focusing
  • Proteome (analysis, metabolism)
  • Proteomics (methods)
  • Water

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