fraction Protocols

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

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 Organelle Protocols: Nucleus Protocols

Cajal Body Isolation Protocol. Protocol includes: Sonication, Removal Nucleoi, Gradient One, Gradient two, Concentration and final enrichment of cajal bodies. Also includes: Making 2.55M sucrose stock and Analysis of the enriched Cajal body fraction. - [Read Cajal Body Isolation Protocol]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol for the isolation of genomic and viral DNA from lymphocytes. Includes: Viral DNA in cytosolic fraction and Viral DNA in nuclear fraction —integrated. - [Read Isolation of Genomic and Viral DNA from Lymphocytes Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations (protocol may be found at www.methods.info). Reduction of the amount of microbeads in comparison to Miltenyi protocol reduces the costs of the experiment. - [Read Isolation of Monocytes from Enriched PBMCs using CD14 Magnetic Beads Protocol]

Category: Cell Biology and Cell Culture

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations. Reduction of the amount of microbeads reduces the costs of the experiment. - [Read Isolation of monocytes from monocyte enriched PBMC fraction using CD14]

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: RNAi Technology Protocols: RNAi Drosophila Protocols

siRNAs produced upon the addition of dsRNA to Drosophila embryo extract are enriched in a micrococcus-nuclease-resistant fraction.  After proteinase K treatment and dephosphorylation with calf intestinal phosphatase, these siRNAs mediate efficient RNAi in vitro. - [Read Preparation of siRNAs from Drosophila Embryo Extracts 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 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 Biology and Cell Culture

Protocol for the recovery of a highly viable fraction for use in fertilization. Protocol uses an iodinated density gradient medium to adjust the density of the raw ejaculate to approx 1.170 g/ml by mixing with a high density medium (ρ >1.26 g/ml) and loading it beneath a discontinuous gradient. - [Read Purification of viable bovine spermatozoa of normal morphology.]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Sorting Protocols

Describes how FACSort can be used to enrich for transfected mouse cells expressing high levels of the human thrombin receptor. The sorted fraction can then be cultured in vivo and reanalyzed 12 days later to show that it remains enriched for thrombin receptor-expressing cells. - [Read Sorting Transfected Cells Based on Gene Expression, Followed by Culture in Vivo]