The preparation of subcellular organelles from mouse liver in self-generated gradients of iodixanol.
This paper reports the use of a new density gradient compound, Iodixanol, for the resolution of the major organelles from mouse liver. A major advantage of Iodixanol over other iodinated density gradient media is its ready ability to form self-generated gradients. Gradient-forming conditions have been modulated to provide optimal recoveries of Golgi membranes, lysosomes, mitochondria, and peroxisomes. The organelles were isolated in high yield (80-90% of gradient input) and high purity. Nycodenz and Iodixanol were compared using preformed gradients. Iodixanol provided resolution superior to that of Nycodenz, notably of peroxisomes and mitochondria and the separation of lysosomes from endoplasmic reticulum. Because Iodixanol does not interfere significantly with marker enzyme activities, gradient fractions can be analyzed without removal of the gradient medium.
Department of Biology, University of Essex, Colchester, United Kingdom.
J
J
Graham
T
T
Ford
D
D
Rickwood
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D002501:Centrifugation, Zonal / Q000379:methods; D003287:Contrast Media; D004798:Enzymes / Q000378:metabolism; D006056:Golgi Apparatus / Q000648:ultrastructure; D007202:Indicators and Reagents; D007472:Iohexol; D008099:Liver / Q000648:ultrastructure*; D008247:Lysosomes / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D008830:Microbodies / Q000648:ultrastructure; D008930:Mitochondria, Liver / Q000648:ultrastructure; D015388:Organelles / Q000648:ultrastructure*; D014283:Triiodobenzoic Acids; D014316:Tritium; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D015415:Biomarkers; D003287:Contrast Media; D004798:Enzymes; D007202:Indicators and Reagents; D014283:Triiodobenzoic Acids; D014316:Tritium; D014531:Uridine Diphosphate Galactose; D007472:Iohexol; C044834:iodixanol