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 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.
Srinivas
Kitambi Satish
KS
Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai-600 020, India.
Chandrasekar
Gayathri
G
Srivastava
Renu
R
Puvanakrishnan
Rengarajulu
R
D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods*; D003579:Cytochrome Reductases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology*
D013395:Sucrose; D005969:Glutamate Dehydrogenase; D003579:Cytochrome Reductases