separation Protocols

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science. - [Read Blue-Native PAGE in Plants: A Tool in Analysis of Protein-Protein Interactions]

Category: Immunology: Mononuclear Cell Isolation Protocols

Protocol for enrichment of PBMCs with monocytes. The cell suspension obtained after this protocol contains 40-70% monocytes. This cell suspension is than used for positive or negative MACS separation. - [Read Enrichment of PBMCs with Monocytes Protocol]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols

Protocol for extraction and purification of total RNA using TRIzol OR TRI reagent. Includes: Homogenization for Cell Suspensions; Phase Separation; RNA Precipitation; RNA Wash;  Redissolving the RNA; Determination of RNA Concentration and Purity; Preparation of Rnase-free water. - [Read Extraction and Purification of Total RNA using TRIzol OR TRI Reagent Protocol]

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

Questions and answers about cell sorting. Includes: When should I use fluorescence activated cell sorting over bulk separation methods like panning or magnetic bead separations? Will my cells be harmed by the sorting process? How many cells do I need to prepare to recover 1 X 106 of a population that comprises 10% of the cells? Are there ways to improve sort recovery? etc... - [Read FAQs About Cell Sorting]

Category: Plant Biology Protocols

Protocol for the fixation and seperation of Arabidopsis leaf cells. Includes: Sample Preparation; Slide Preparation. - [Read Fixation and Separation of Arabidopsis Leaf Cells Protocol]

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: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Protocol for Immunoblot. Includes: Staining and Laser Capture Microdissection; Protein Separation by Polyacrylamide Gel Electrophoresis; Electrophoretic Transfer To a Membrane (Nylon, PVDF or Nitrocellulose); Primary and Secondary Antibody Incubations; Visualization. - [Read Immunoblot Protocol]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol for the isolation of infectious HIV-1. Includes: HIV-1 capture strategy; Protocol for the isolation of HIV-1 virions; Magnetic labeling; Magnetic separation; Elution option A for virion lysate; Elution option B for intact virions. - [Read Isolation of Infectious HIV-1 Protocol]

Category: Microsphere Analysis Protocols

The role of microspheres in these screens is similar to their traditional role in immunoassays, namely as a solid phase to either enhance detection, separation, or both. The predominance of radioactive assays in high-throughput screening, along with the desire to find alternative means of detection, have led to research on substituting alternative fluorescent technologies. - [Read Microspheres for High-Throughput Screening Assays]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

The protocol consists of a method for the generation of cytoplasmic extracts from mammalian cells (in this case, 293T cells) without the disruption of polyribosomes, the separation of ribosomal components and polyribosomes by sucrose gradient centrifugation, the isolation of mRNA from these fractions, and detection of mRNA by Northern blot analysis. - [Read Northern Blot Analysis of mRNA From Mammalian Polyribosomes Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Provides an overview of terminology and echniques unique to flow ytometry and is divided into two sections. The first section discusses technical aspects of flow cytometry which apply primarily to the instrumentation-oriented flow cytometry phase. The second section discusses electronic cell separation using flow cytometry. - [Read Overview of Flow Cytometry]

Category: Immunology: B Cell Protocols

Procedure permits the isolation of at least 5 µg of total RNA from a sample of purified mouse splenic B lymphocytes. The quality of the RNA is assessed by separation of an aliquot through 1% agarose and staining with ethidium bromide as described in AfCS protocol Visualization of RNA Preparations on 1% Agarose Gels. The isolated RNA is used for analysis of gene expression by microarray technology. analysis of gene expression by microarray technology. - [Read Preparation of B-Lymphocyte RNA for Microarray Analysis Protocol]

Category: Antibody Protocols: Antibody Production Protocols

Polyclonal antibodies can be isolated from animal plasma or serum using the procedure described in this protocol. The Gradiflow BF400 instrument has two liquid streams that circulate through a separation cartridge positioned between two electrodes and composed of three hydrogel polyacrylamide membranes, which define the channels for the two sample streams. The central membrane forms a physical barrier between the two streams. - [Read Preparation of Polyclonal Antibodies from Plasma or Serum Using the Gradiflow BF400]

Category: Protein Protocols: Protein Extraction Protocols

Protocol for Protein Extraction Using Proteomics. Extraction of proteins from plant cells that are rich in compounds that interfere with the 2-Dimensional electrophoretic separation methods such as salts, organic acids, phenolics, pigments, terpenes, among others. A common protocol used in our lab for extraction proteins from plant tissues consists in the homogenization of mortar-grounded material in liquid nitrogen with an extraction buffer. - [Read Protocol for Protein Extraction Using Proteomics]

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 Biology and Cell Culture

Method separates monocytes and lymphocytes on the basis of density and size. - [Read Separation of monocytes from human leukocyte-rich plasma by flotation.]

Category: Cell Biology and Cell Culture

This protocol describes how to isolate human platelets from whole blood. Isolated platelets are used for static adhesion assays, for flow chamber assays, flow cytometry measurements, etc. - [Read Separation of Platelets from Whole Blood]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Separation of RNAs according to size is the first stage in northern blotting and hybridization.  The method described in this protocol uses glyoxal to denature the RNA, ethidium bromide to stain it, and agarose gel electrophoresis to separate the resulting glyoxal-RNA-ethidium adducts. - [Read Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Separation of RNAs according to size is the first stage in northern blotting and hybridization.  The method described in this protocol uses formaldehyde to denature the RNA, ethidium bromide to stain it, and electrophoresis through agarose gels containing 2.2 M formamide to separate the resulting formaldehyde-RNA-ethidium adducts. - [Read Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formalde]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol describes the first stages of Southern blotting: digestion of genomic DNA with one or more restriction enzymes, separation of the resulting fragments by electrophoresis through an agarose gel, and transfer of the denatured fragments to a membrane by downward capillary transfer. - [Read Southern Blotting: Capillary Transfer of DNA to Membranes Protocol]

Category: Immunology: Immunoassays

Protocol for steroid radioimmunoassay. Includes: SOLVENT DISTILLATION; PREPARATION OF PLASMA SAMPLES; EXTRACTION OF STEROIDS AND COLUMN PACKING; COLUMN CHROMATOGRAPHY; RADIOIMMUNOASSAY; SEPARATION OF BOUND AND FREE COUNTS; DIRECT ASSAYS; SHORT COLUMN CHROMATOGRAPHY. - [Read Steroid Radioimmunoassay Protocol]

Category: Microscopy Protocols: Optical Microscopy Protocols

TIRM is a optical technique for monitoring the instantaneous separation distance between a microscopic sphere & a flat plate. Changes in distance as small as 1 nm can be detected. Includes information on: Scattering Intensity I is Related to Elevation h ; apparatus. - [Read Total Internal Reflection Microscopy]