Subcellular Fractionation Protocols

Protocol for percoll gradient. Includes: Forming gradient, Removing percoll and reagents. - [Read Percoll Gradient Protocol]

Protocol for cell disruption and subcellular fractionation. Includes: Nitrogen Bomb, Fractionation and Reagents. - [Read Cell Disruption and Subcellular Fractionation 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. - [Read Preparation of Crude Subcellular Fractions by Differential Centrifugation Protocol]

Protocol describes systems in which the membranes are allowed to float through a density gradient from a dense load zone. This is an ideal format. - [Read A Self-generated Gradient to Discriminate a Cytosolic or Membrane Location of Large Protein Complex]

This protocol describes nuclear and cytoplasmic fractionation of tissue culture cells and a method for Western blot detection of proteins using the Odyssey Infrared Imaging System. This protocol was used to detect expression of the "small" Tap protein in 293T, HeLa and COS cells. The Odyssey system has several advantages over the more widely used chemiluminescent detection methods. - [Read Western Blot Analysis of Sub-Cellular Fractionated Samples Using the Odyssey Infrared Imaging System]

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. - [Read Fractionation of Vacuolar and Subvacuolar vesicles and Vacuole and Cytoplasm-to-Vacuole Targeting]

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. - [Read Fractionation of Human Erythrocytes (Normal or Sickle) and Reticulocytes in Discontinuous Iodixanol]

Protocol for isolation of dendritic cell derived exosomes. - [Read Dendritic Cell Derived-Exosomes]

Acidocalcisomes, the dense acidic calcium-storing organelles, which were originally identified in Trypanosoma cruzi, have no parallels in mammalian cells. They thus represent a unique functional characteristic, not shared by the host and hence offer an important potential target for chemotherapy of Chagas disease. - [Read Fractionation of Acidocalcisomes and Other Organelles from Trypanosoma, Leishmania, Chlamydomonas]

This protocol is employed for the purification of a population of muscle-derived cells from skeletal muscle of C57Bl/6 animals. The resulting preparation is a mixture of many cell types including satellite cells (a.k.a., muscle progenitor cells) and hematopoietically active muscle-derived cells. - [Read Protocol for the Isolation of a Heterogenous Muscle-Derived Cell Population]

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. - [Read Subfractionation of Low-Density Lipoprotein (LDL)]

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. - [Read Purification of Oocyst Walls and Sporocysts from Toxoplasma gondii Protocol]