A novel protocol for the subcellular fractionation of C3A hepatoma cells using sucrose density gradient centrifugation

In this paper, we describe a method to obtain a relatively pure mitochondrial and microsomal fractions by subcellular fractionation of human hepatoma cell line C3A using sucrose as the hypoosmotic medium. The cells were subjected to osmotic stress with sucrose and homogenized. Osmolarity was then re...

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Bibliographic Details
Published in:Journal of biochemical and biophysical methods 2004-07, Vol.60 (1), p.23-27
Main Authors: Srinivas, Kitambi Satish, Chandrasekar, Gayathri, Srivastava, Renu, Puvanakrishnan, Rengarajulu
Format: Article
Language:English
Subjects:
Blotting, Western
C3A cells
Carcinoma, Hepatocellular - metabolism
Cell Fractionation - methods
Cell Line, Tumor
Cell Nucleus - metabolism
Centrifugation, Density Gradient - methods
Cytochrome Reductases - metabolism
Electrophoresis, Polyacrylamide Gel
Glutamate Dehydrogenase - metabolism
Humans
Liver - metabolism
Liver Neoplasms - metabolism
Microsomes - metabolism
Microsomes, Liver - metabolism
Mitochondria - metabolism
Subcellular fractionation
Subcellular Fractions - metabolism
Sucrose - pharmacology
Sucrose density gradient
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Summary:In this paper, we describe a method to obtain a relatively pure mitochondrial and microsomal fractions by subcellular fractionation of human hepatoma cell line C3A using sucrose as the hypoosmotic medium. The cells were subjected to osmotic stress with sucrose and homogenized. Osmolarity was then restored to the cells and the organelles were separated by density gradient centrifugation. The protein profiles were examined by SDS-PAGE and the purity was analysed by marker enzymes and Western blotting. Our results indicate a good separation of mitochondrial and microsomal fractions from human hepatoma C3A cells.
ISSN:0165-022X
1872-857X
DOI:10.1016/j.jbbm.2004.04.011