期刊
ANALYTICAL BIOCHEMISTRY
卷 394, 期 2, 页码 249-258出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.07.026
关键词
Differential centrifugation; Disruption; Isolation; Mitochondria; Proteomics; Proteome; Purity; Sonication
资金
- Thailand Research Fund
- Commission on Higher Education, Mahidol University
- National Research Council of Thailand
- National Center for Genetic Engineering and Biotechnology
- Siriraj Graduate Thesis Scholarship
One of the most crucial steps in mitochondrial isolation is disruption of intact cells to denude intracellular organelles, but the yield and purity of different disruption protocols have not been well addressed. In the present study, MDCK cells were disrupted by mechanical ( sonication and homogenization), physical ( repeated freeze/thaw cycles and hypoosmotic burst), and chemical ( using Triton X-100, NP-40, or CHAPS) methods. Efficacy of cell disruption was evaluated by trypan blue staining and mitochondria were subsequently isolated by standardized differential centrifugation. The yield of isolation was also determined by measuring protein concentrations, whereas the purity was examined by Janus green B staining, Western blot analyses of markers for mitochondria (COX-4) and other subcellular organelles/locales (i.e., nucleus, cytoplasm, endoplasmic reticulum, and lysosome), transmission electron microscopy, two-dimensional electrophoresis, and Q-TOF MS and/or MS/MS analyses. Our data demonstrated that sonication is the method of choice for disruption of cells prior to mitochondrial isolation for proteome analysis. (C) 2009 Elsevier Inc. All rights reserved.
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