Fractionation of Human Erythrocytes (Normal or Sickle) and Reticulocytes in Discontinuous Iodixanol A number of density gradient strategies have been developed for the fractionation of human erythrocytes according to their age. As the cells age, so their density tends to increase; reticulocytes therefore tend to have the lowest densities. Reticulocytes have frequently been partially purified on discontinuous gradients of arabinogalactan; the actual density range being quite varied, from quite broad ones.
Fractionation of Vacuolar and Subvacuolar vesicles and Vacuole and Cytoplasm-to-Vacuole Targeting Fractionation of (a) vacuolar and subvacuolar vesicles and (b) vacuole and cytoplasm-to-vacuole targeting (Cvt) vesicles from yeast spheroplasts in a pre-formed discontinuous iodixanol gradients. Protocol includes: Formation of yeast spheroplasts; Isolation and vesiculation of the vacuoles; Separation of the vacuolar and subvacuolar vesicles; Separation of vacuoles and Cvt vesicles from a yeast spheroplast lysate.
Preparation of Crude Subcellular Fractions by Differential Centrifugation Protocol The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. This protocol describes the use of differential centrifugation to fractionate a mammalian liver
homogenate but similar methods should be applicable to all mammalian tissues and cultured cells.
Purification of Oocyst Walls and Sporocysts from Toxoplasma gondii Protocol Although Percoll gradients were able to provide a purified sporocyst fraction, because these particles do not all band in a discrete manner in such gradients, they were unable to provide a simultaneous isolation of a pure oocyst wall fraction. Gradients formed from this protocol on the other hand are able to provide purified sporocysts and oocyst walls in the same gradient.
Subfractionation of Low-Density Lipoprotein (LDL) In the routine method described in this protocol, chylomicron-free plasma is adjusted to 12% (w/v) iodixanol and the sample, essentially fills an approx 3 ml tube for a near-vertical rotor. During the centrifugation VLDL, LDL and HDL particles and also plasma proteins migrate from all parts of the sample to their final buoyant density banding position in the self-forming density gradient.