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 organelle s from the rat liver and subsequent proteome analysis of subcellular fracti ons. To prepare two-dimensional electrophoresis (2-D PAGE) profiles of tiss ue organelles, we performed onestep 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 cen trifuge tube by freezing-thawing overnight at -20 degreesC and at room temp erature for a few hours without the initial centrifugation procedure. The s hape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined usin g one-dimensional sodium dodecyl sulfate (SDS)-PAGE. The organelles were co nfirmed using Western blotting. Our results indicate that our procedure pro vides a simple method for the separation of organelle fractions from the ra t liver tissue.