axis Protocols

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

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]

Category: Protein Protocols: Protein Trafficking

Protocol is based on methods for the resolution of GLUT4 containing vesicles and the identification of phosphoinositide kinase containing vesicles in 3T3-L1 adipocytes. They may have a wider application to any low-medium density membranes. Protocol incorporates the strategy of using a low density microsome fraction as the gradient input, commonly used in GLUT 4 studies that may have a wider application to other investigations. - [Read Analysis of Membrane Trafficking and Intracellular Signaling in Self-Generated Iodixanol Gradients]

Category: Cell Biology and Cell Culture

Techniques on how to create gradients of iodixanol for the fractionation of mammalian cells. These gradients can be generated as pre-formed discontinuous or continuous gradients. These gradients are invariably run in swinging-bucket rotors in low-speed centrifuges. - [Read C2 Preparation of pre-formed iodixanol gradients for mammalian cells.]

Category: Cell Biology and Cell Culture

OptiPrep Application sheet C14 describes procedures for determining the density and sedimenting properties of any cell (of any size or density) using either a continuous or discontinuous gradient of iodixanol. This Application Sheet describes procedures aimed at isolating specifically a relatively low-density cell fraction from any tissue. - [Read C25 Isolation from spleen, thymus, pancreas, alveolar tissue and other tissues]

Category: Cell Biology and Cell Culture

The protocol presented in this Application Sheet uses an alternative strategy to sedimentation on to a density barrier, that is to adjust the density of whole blood to a value just greater than the cells of interest and allow them to float to the surface. - [Read C8 Isolation of bovine peripheral blood mononuclear cells by flotation.]

Category: Cell Biology and Cell Culture

Cell fractionation techniques are presented for the design of gradient systems for separating one or more cell types from lavages of body cavities or from mechanically or enzymically-dissociated tissues. Includes: Preparation of cell suspension for gradient loading; Fractionation by buoyant density; Fractionation on the basis of cell size. - [Read Cell Fractionation of a Mixed Population of Cells]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

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]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

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. - [Read Fractionation of Golgi, ER, TGN and Other Membrane Compartments in Pre-Formed Iodixanol Gradients]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

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]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

Generally in iodixanol gradients the density of organelles decreases in the series: peroxisomes, mitochondria, lysosomes, ER, Golgi, although in Dictyostelium discoideum, the lysosomes are denser than the mitochondria. Iodixanol gradients can usually provide satisfactory resolution of all these membrane particles although it may be necessary to modulate either the gradient or centrifugation parameters in order to optimize a particular separation. - [Read Fractionation of Mitochondria, Lysosomes, Peroxisomes, ER and Golgi in Pre-formed Iodixanol Gradient]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

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]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

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. - [Read Fractionation of Yeast Membranes in Pre-Formed Continuous Iodixanol Gradients]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

A discontinuous gradient of iodixanol is used in which the crude mitochondrial fraction is layered beneath the preformed gradient. In this protocol the osmolality of the gradient is approx 600 mOsm and the crude mitochondrial fraction is loaded in 40% iodixanol rather than 50% iodixanol. - [Read Isolation of Yeast Mitochondria in aPre-Formed Iodixanol Gradient]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope when three planes along the z-axis of the cell are acquired. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP & Bright Field—Three Z Axis]

Category: Cell Biology and Cell Culture

Purification protocols of the viruses: HIV-1, Lassa virus, oncornavirus and other retroviruses. Protocol uses an iodixanol gradient in a sedimentation velocity mode to purifyHIV-1 virions without affecting the infectivity of the virus. In rate-zonal iodixanol gradients the HIV-1 was effectively separated both from Vif and from the microvesicles. - [Read M5 Velocity (rate zonal) gradients for purification and assembly analysis of viruses.]

Category: Cell Biology and Cell Culture

Cell fractionation protocol to yield intact plant protoplasts. Technique used to purify protoplasts from the grass Glyceria fluitans. Protocol includes: Determination of leaf osmolality; Sterilization. - [Read Plant Cell Purification of Intact Plant Protoplasts]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

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]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

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. - [Read Purification of Caveolae Membranes from a Plasma Membrane Fraction of Cultured Cells and Tissues]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

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. - [Read Purification of Golgi Membranes from a Light Mitochondrial Fraction in a Self-Generated Gradient]

Category: Cell Biology and Cell Culture

The separation depends on the use of a hyperosmotic density barrier to separate the two types of leukocyte. - [Read Purification of leukocyte fractions from rat peritoneal exudates by density barrier sedimentation.]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

This protocol describes a discontinuous gradient, which resolves the mitochondria from both lighter and denser organelles. Because the centrifugation is carried out for 4 h, diffusion will create a partially continuous gradient and this probably contributes to the resolution of the mitochondria from the lighter lysosomes. - [Read Purification of Mammalian Liver Mitochondria by Flotation Through a Pre-formed Discontinuous Iodixan]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

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]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Peroxisome Isolation Protocols

Peroxisomes can be purified in self-generated iodixanol gradients in high yield (80-90%) with no detectable contamination from any other organelle. In iodixanol peroxisomes are the densest of the major subcellular organelles (ρ = 1.18-1.20 g/ml) present in the light mitochondrial fraction from mammalian tissues and cells. - [Read Purification of Peroxisomes in a Self-Generated Gradient]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Peroxisome Isolation Protocols

Peroxisomes can be purified in iodixanol gradients in high yield (80-90%) with no detectable contamination from any other organelle. This is a property unique to iodixanol because the densities of other organelles, particularly that of mitochondria (approx ρ = 1.14 g/ml) and endoplasmic reticulum (approx ρ = 1.13 g/ml) are much lower than that of peroxisomes (approx ρ = 1.18 g/ml). - [Read Purification of Peroxisomes using a Density Barrier in a Swinging-Bucket Rotor]

Category: Viral Protocols

Protocol for the purification of recombinant adeno-associated virus (rAAV) and parvovirus in pre-formed iodixanol gradients. - [Read Purification of Recombinant Adeno-Associated Virus (rAAV) and Parovirus Protocol]