Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient Centrifugation

Peroxisome purification depends on a two-step procedure: differential centrifugation to prepare a light mitochondrial fraction and fractionation on a density-gradient medium preferably iodixanol or Nycodenz, to isolate the peroxisome enriched fraction. The iodixanol gradient may be a preformed continuous gradient or a self-generating gradient. Alternatively a continuous Nycodenz gradient or a simple Nycodenz barrier may be used for the second step. The unit contains protocols for peroxisome isolation from rat liver, tissue culture cells (HepG2 cells), and yeast spheroplasts. The extent of endoplasmic reticulum contamination of the prep can be assessed using an assay for the marker enzyme NADPH-cytochrome creductase.