gradient Protocols

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

A solution containing plasmid DNA, saturating amounts of ethidium bromide, and CsCl (44% w/v) is layered between two solutions of lesser (35% w/v CsCl) and greater density (59% w/v CsCl).  During centrifugation to equilibrium, the closed circular plasmid DNA and linear DNAs form bands at different densities. - [Read Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradient]

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: 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: Mitochondria Isolation Protocols

A procedure developed to purify simultaneously peroxisomes and mitochondria from spinach (Spirnacia olercea L.) leaf under isoosmotic and low viscosity conditions. This method involves differential centrifugation and density gradient centrifugation on four layers of Percoll. - [Read Purification of Peroxisomes and Mitochondria from Spinach Leaf by Percoll Gradient Centrifugation]

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

A procedure developed to purify simultaneously peroxisomes and mitochondria from spinach (Spirnacia olercea L.) leaf under isoosmotic and low viscosity conditions. This method involves differential centrifugation and density gradient centrifugation on four layers of Percoll. - [Read Purification of Peroxisomes and Mitochondria from Spinach Leaf by Percoll Gradient Centrifugation]

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 Organelle Protocols: Subcellular Fractionation Protocols

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)]

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

Protocol for sucrose gradient. - [Read Sucrose Gradient Protocol]

Category: Immunology: Chemotaxis

TransWell Chemotaxis protocol. trans-well chemotaxis method from bioprotocol. The protocol is a method for studying migration towards a concentration gradient of chemoattractant of leukocytes (neutrophils, monocytes and lymphocytes) or other migratory cells. An upper chamber containing a suspension of cells is separated by a membrane from a lower chamber containing medium with chemoattractant. Chemotaxis of the cells from the upper chamber into the lower chamber can be quantified. - [Read TransWell Chemotaxis Protocol.]