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Isolation of Intact Organelles from cultured mammalian cells and tissue
Author(s) -
Ignacio Rei,
Loeb J.,
Benton B.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1039
Subject(s) - organelle , microbiology and biotechnology , cell fractionation , proteomics , proteome , biology , organoid , lysis buffer , western blot , differential centrifugation , peroxisome , lysis , chemistry , biochemistry , gene , enzyme
There is considerable effort in the area of proteomics to map and catalog the unique proteomic profile of each organelle. Subcellular fractionation enables the simplification of complex protein mixtures and thereby facilitates this type of proteomic analysis. Pierce developed three organelle enrichment kits for lysosomes, peroxisomes, and nuclei that enable enrichment of intact organelles from cells and/or tissue. Mechanical lysis was employed to effectively lyse cells and tissue, and density gradient centrifugation was used as a method to separate each organelle of interest. The procedure was optimized for diverse cell lines, including A431, HeLa, and HepG2 as well as multiple tissues from Sprague‐Dawley rats including liver, heart, and kidney. The purity of the isolated organelles was assessed by Western blot analysis using antibodies against specific marker proteins on targeted organelles. Benchmarking was performed against commercially available kits and a higher yield with significantly less contamination was demonstrated with each of the Pierce protocols. Isolated organelles may be used for a number of downstream applications, including 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies.