fractionation Protocols

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 Organelle Protocols: Subcellular Fractionation Protocols

Protocol for cell disruption and subcellular fractionation. Includes: Nitrogen Bomb, Fractionation and Reagents. - [Read Cell Disruption and Subcellular Fractionation Protocol]

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 Biology and Cell Culture

Cell fractionation protocol using low speed seperation. - [Read Cell Fractionation protocol Usingt Low Speed Separaton of Cells]

Category: Cell Organelle Protocols: Nucleus Protocols

Protocol for chromatin isolation by small-scale biochemical fractionation. - [Read Chromatin Isolation by Small-Scale Biochemical Fractionation Protocol]

Category: DNA Protocols: cDNA Library Protocols

Protocol for construction of cDNA library, stage 5: Fractionation of cDNA by Gel Filtration through Sepharose CL-4B. - [Read Construction of cDNA Libraries Stage 5: Fractionation of cDNA by Gel Filtration through Sepharose]

Category: Cell Biology and Cell Culture

Cell fractionation of cellular components using Percoll a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. Percoll becomes a simple matter to establish a linear density gradient. Organelle separations are much easier to accomplish on Percoll density gradients than on sucrose gradients. - [Read Equilibrium Density Gradient Percoll Protocol]

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: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

Protocol describes a method for performing isoelectric fractionation of a maize embryo sample using a multicompartment electrolyzer(MCE). This prefractionation of proteins having pIs within a certain pH interval is essential for allowing high loads of protein to be resolved on narrow and ultra-narrow immobilized pH gradients used in 2D electrophoresis. The isoelectric membranes in the MCE act like isoelectric traps capturing all the protein species having pIs encompassing the pI value of each... - [Read Fractionation of Maize Embryo Proteins for 2-D Gel Electrophoresis Using Multicompartment Electrolyz]

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: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

Protocol describes the purification of mouse T cells, B cells, and T cell subsets using magnetic bead separation. Isolation of cell subsets using magnetic beads is quick, simple, and reliable and can result in high yields of very pure cells. - [Read Fractionation of T and B Cells Using Magnetic Beads Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocol describes the purification of mouse T cells, B cells, and T cell subsets using magnetic bead separation. Isolation of cell subsets using magnetic beads is quick, simple, and reliable and can result in high yields of very pure cells. - [Read Fractionation of T and B Cells Using Magnetic Beads Protocol]

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: Molecular Imaging: Immunofluorescence Microscopy

Indirect immunofluorescence with preextraction (in situ cell fractionation). Raffaella Di Micco and fabrizio d'Adda di fagagna. - [Read Indirect immunofluorescence with preextraction (in situ cell fractionation)]

Category: Immunology: Immunofluorescence Protocols

The study of the activation of DNA damage response factors at the single cell level relies on the observation of their accumulation (or the accumulation of their activated forms) in nuclear foci by plain immunofluorescence techniques. - [Read Indirect Immunofluorescence with Preextraction (In Situ Cell Fractionation) Protocol]

Category: Lipid Protocols

Lipoprotein isolation protocol using insect hemolymph. Includes: Bleeding and centrifugation; Fractionation, refractometry and dialysis; Lyophilization. - [Read Lipoprotein Isolation Protocol]

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

To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. - [Read Peroxisomes in Saccharomyces cerevisiae]

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: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes a method to prepare and fractionate Drosophila embryo extracts.  These extracts can be used for the in vitro characterization of RNAi. - [Read Preparation and Fractionation of Drosophila Embryo Extracts for the In Vitro Characterization of RNA]

Category: Protein Protocols: Protein Purification and Identification

Protein Purification: Assays, Specific Activity, Initial Fractionation. Blaber Lecture. - [Read Protein Purification: Assays, Specific Activity, Initial Fractionation]

Category: Protein Protocols: Protein Extraction From Tissue

Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs / tissue - [Read Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomic]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

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]