One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles.
In the present study, we describe a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two-dimensional electrophoresis (2-D PAGE) profiles of tissue organelles, we performed one-step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing-thawing overnight at -20 degrees C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one-dimensional sodium dodecyl sulfate (SDS)-PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.
Murayama
K
K
Division of Biochemical Analysis, Juntendo University School of Medicine, Tokyo, Japan. murayama@med.juntendo.ac.jp
Fujimura
T
T
Morita
M
M
Shindo
N
N
D000818:Animals; D015415:Biomarkers; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005615:Freezing; D007472:Iohexol; D008099:Liver / Q000737:chemistry*; D008928:Mitochondria / Q000201:enzymology; D015388:Organelles / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000737:chemistry*; D013385:Succinate Dehydrogenase / Q000032:analysis; D014461:Ultracentrifugation
D015415:Biomarkers; D007472:Iohexol; D013385:Succinate Dehydrogenase