Isolation of intestinal cell peroxisomes by rate-dependent banding in a vertical rotor.
The preparation of peroxisome-enriched fractions from mouse intestinal epithelial cells is possible by ultracentrifugation using the vertical rotor under rate-zonal conditions. The run time is short which results in a low total centrifugal effect to which these fragile organelles are subjected. The banding of peroxisomes occurs in the central region of the sucrose gradient whereas the mitochondria band in the lower regions. Good separation from the mitochondria is achieved with contaminants resulting from brush-border fragments and endoplasmic reticulum. Comparisons are made with preparations using a swing-out rotor and a zonal rotor. Electron microscope examination of the preparations confirms the presence of diaminobenzidine-positive particles which resemble the peroxisomes seen in intact cells. In addition, it has been shown that density gradients can be constructed which prevent large particles from impacting and causing wall effects.
Gianello
R
R
Monash University, Clayton, Victoria, Australia.
Nayudu
R
R
D000818:Animals; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D004848:Epithelium / Q000648:ultrastructure; D007413:Intestinal Mucosa / Q000648:ultrastructure*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008830:Microbodies / Q000648:ultrastructure; D008854:Microscopy, Electron; D014461:Ultracentrifugation / Q000295:instrumentation*