Fractionation of Golgi, ER, TGN and Other Membrane Compartments in Pre-Formed Iodixanol Gradients The protocol described in this protocol has been used principally for analyzing the Golgi, endoplasmic reticulum and trans-Golgi network but markers for other compartments (e.g.
ERGIC and endosomes) have also been analyzed. Modifications either to the gradient density range or the centrifugation conditions influence the ability of the gradient to resolve multiple compartments.
Fractionation of Yeast Membranes in Pre-Formed Continuous Iodixanol Gradients This protocol is concerned with the use of iodixanol gradients in an analytical mode to study the membrane localization of a particular protein or function. Continuous gradients are best suited to this task. One of the protocols described in this protocol starts with a discontinuous gradient, but since the gradient is centrifuged at 174,000g for 16 h it will become continuous by diffusion.
Purification of Caveolae Membranes from a Plasma Membrane Fraction of Cultured Cells and Tissues The first part of the isolation procedure is a flotation through a continuous iodixanol gradient; this gradient is essentially a resolving gradient in which the caveolin-rich vesicles are concentrated in the top third of the gradient, while the predominantly caveolin-poor vesicles band in denser regions. A second discontinuous gradient is essentially a concentration gradient to band the caveolin-rich vesicles sharply at an interface.
Purification of Golgi Membranes from a Light Mitochondrial Fraction in a Self-Generated Gradient This protocol uses a "light mitochondrial" pellet from a mammalian liver homogenate. The gradient thus has to resolve a variety of denser components (peroxisomes, lysosomes, mitochondria) from the Golgi membranes, which have a low density in iodixanol (1.06-1.09 g/ml) [1]. The protocol is
specifically tailored to the purification of Golgi membranes from this pellet and is unsuitable for the isolation or analysis of other organelles present in the light mitochondrial fraction.
