PMID: 16522859Mar 9, 2006Paper

Proteomic changes in bovine heart mitochondria with age: using a novel technique for organelle separation and enrichment

Journal of Biomolecular Techniques : JBT
Ajay N KiriMarcus J Horn

Abstract

Separation and enrichment of organelles from complex biological mixtures are important for proteomic analysis. Two widely used current standard techniques to isolate individual organelles include differential and density-gradient centrifugation. Although these techniques have proven useful for processing small volumes of sample, multiple rounds of centrifugation are required when performing a large-scale purification. In this report, we have introduced a novel technique: continuous-flow ultracentrifugation using a sucrose gradient to separate, accumulate, and highly enrich bovine heart mitochondria in one step. To demonstrate the advantage of the technique, mitochondrial proteins from two different bovine hearts (3-8 mo and 18-30 mo old) were examined. For each age group, 100 g of bovine heart tissue were homogenized by a blending procedure. After removal of the nuclei, the entire remaining homogenate was loaded onto a proteomics continuous-flow ultracentrifuge to separate and enrich the organelles. Fractions were collected and mitochondria-enriched fractions were identified by Western blot analysis. To study the protein profile changes with aging in the mitochondrial proteome, the mitochondria-enriched fractions were applied t...Continue Reading

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