Comparative proteomics: Characterization of a two-dimensional gel electrophoresis system to study the effect of aging on mitochondrial proteins

Jinsook Chang, Holly Van Remmen, John Cornell, Arlan Richardson, Walter F. Ward

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

To study the effect of aging and anti-aging strategies on mitochondria, we have characterized a two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system to analyze the profile of mitochondrial proteins. We have optimized the separation of proteins by 2-D PAGE and established the linearity and reproducibility of the system with mitochondria isolated from skeletal muscle of mice. Using total mitochondria protein ranging from 10 to 200 μg, we found that 74% of the proteins resolved by 2-D PAGE had coefficient of determination (R2) values greater than 0.8, showing a linear increase in fluorescence with increasing protein concentration. The coefficient of variation (CV) was less than 50% for at least 93% of the 424 spots analyzed for both gel-to-gel variance and animal-to-animal variance. Using mitochondrial protein fractions prepared from skeletal muscle of 18-month-old mice, we show that 10 animals will be sufficient to detect a 100% difference in the 97% (i.e. 505) of the proteins resolved by 2-D PAGE. Thus, 2-D PAGE provides a sensitive and reliable technique for analysis of protein expression in mitochondria.

Original languageEnglish (US)
Pages (from-to)33-41
Number of pages9
JournalMechanisms of Ageing and Development
Volume124
Issue number1
DOIs
StatePublished - Jan 1 2003

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Keywords

  • 2-D PAGE
  • Aging, skeletal muscle
  • Mitochondria proteins

ASJC Scopus subject areas

  • Aging
  • Developmental Biology

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