کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1175783 961817 2009 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study
چکیده انگلیسی

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 394, Issue 2, 15 November 2009, Pages 249–258
نویسندگان
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