PMID: 16522856Mar 9, 2006Paper

Comparison of Golgi apparatus and endoplasmic reticulum proteins from livers of juvenile and aged rats using a novel technique for separation and enrichment of organelles

Journal of Biomolecular Techniques : JBT
Kate L DrahosMarcus J Horn

Abstract

The broad dynamic range of protein abundances, which can vary from about 10(6) for cells to 10(10) for tissues in complex proteomes, continues to challenge proteomics research. Proteome analysis, in particular organelle proteomics, using current approaches, requires extensive fractionation, separation, and enrichment. Over the years, organelle separation was achieved through the use of differential and density-gradient ultracentrifugation. However, the traditional fixed-volume process is a time-consuming and labor-intensive method, especially with large quantities of sample. Here, we present a novel tool for subcellular fractionation of biologically complex mixtures: continuous-flow ultracentrifugation of tissue homogenates to obtain both organelle separation and extensive organelle enrichment at the same time. In this study, rat liver tissues from two different age groups (3-8 wk and greater than 1 y old) were homogenized by blending. After removing nuclei, the resulting homogenates were further fractionated at the subcellular level by the use of sucrose gradient continuous-flow ultracentrifugation. Each organelle's enriched fractions were identified by Western blot analysis. To study the possible effects of aging on the endop...Continue Reading

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