membranes 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: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, in cultured adherent RAW 264.7 cells, using a 96-well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fluo-3, which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, in cultured adherent RAW 264.7 cells, using a 96- well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fluo-3, which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. Fluorescence for the adherent cells is measured over time by using a bottom read of a 96-well plate, with cells that have been washed. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fluo-3 Protocol]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+], in cultured adherent RAW 264.7 cells, using a 96-well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fura-2, which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fura-2 (with FLEXstation)]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i , in cultured adherent RAW 264.7 cells, using a 96- well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fura-2, which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fura-2 Protocol]

Category: DNA Protocols: DNA Extraction Protocols: Agarose Gel DNA Extraction Protocols

DNA Purification from DEAE Nitrocellulose Membranes Protocol.  Jeff Lawrence - [Read DNA Purification from DEAE Nitrocellulose Membranes Protocol]

Category: Protein Protocols: Western Blot Protocol

ENZYME-ASSISTED IMMUNOELECTROBLOTTING (IEB OR WESTERN BLOTTING. E.P. Rybicki and Maud Purves. Department of Microbiology. Copper Staining of Gels, Indirect enzyme immunoassay, Staining Proteins on Membranes, Blotting Buffer, Staining of proteins in gels - [Read ENZYME-ASSISTED IMMUNOELECTROBLOTTING (IEB OR WESTERN BLOTTING)]

Category: Recombinant Protein Protocols

Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography. Biological Procedures Online - [Read Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affin]

Category: Microscopy Protocols

FM 4-64 is a lipophilic styryl dye and a vital stain: it fluoresces only in living cells, so cells cannot be fixed then stained nor stained then fixed. You must stain and observe living cells. FM 4-64 does not permeate cell membranes but, instead, intercalates into the plasma membrane is then taken into the cells by endocytosis. - [Read FM 4-64 Labeling of Yeast Vacuole Membranes Protocol]

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: 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: Western Blot Protocols: General Western Blot Methods Protocols

The transfer of proteins from a Tris/glycine SDS-polyacrylamide gel to a membrane using a semi-dry method is achieved by placing the gel next to a piece of nitrocellulose filter.  This sandwich is placed directly between two plate electrodes, and the proteins are then transferred from the gel onto the filter. - [Read Immunoblotting: Semi-Dry Electrophoretic Transfer of Proteins from Gels to Membranes Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

Transfer of proteins from a Tris/glycine SDS-polyacrylamide gel to a membrane using the submerged method is achieved by placing the gel next to a piece of nitrocellulose filter, submerging this sandwich in a large volume of transfer buffer in a transfer tank, and running current from one side of the transfer tank to another.  The proteins are then eluted by transferring them from the gel onto the filter. - [Read Immunoblotting: Submerged Electrophoretic Transfer of Proteins from Gels to Membranes Protocol]

Category: Yeast Protocols

Protocol for in vitro endoplasmic reticulum to golgi transport reaction in Yeast. Includes: Preparation of Membranes; One-Stage Reaction; Two-Stage Reaction using Normal Amount of Membranes; Two-Stage Reaction using Low Concentration of Membranes. - [Read In Vitro Endoplasmic Reticulum to Golgi Transport Reaction in Yeast Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol outlines the general procedure and requirements for in vitro translation of CFTR and outlines some assays using in vitro translated product. Core glycosylation of CFTR occurs in the ER. An assay for this processing step requires the addition of microsomal membranes to the basic in vitro translation mixture. This protocol takes this into account. - [Read In vitro Translation Assays for CFTR]

Category: Mouse Protocols: Mouse Embryology Protocols

Isolation of extraembryonic tissues allows one to study normal mouse development as well as the molecular basis of defects which cause fetal death. This protocol describes a method for isolating extraembryonic membranes from pregnant mice. - [Read Isolating Mice Extraembryonic Membranes Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Method of choice when large amounts of mammalian DNA are required, for example, for Southern blotting (Rapid Isolation of Mammalian DNA, Rapid Isolation of Yeast DNA, Southern Blotting: Capillary Transfer of DNA to Membranes) or for construction of genomic libraries in bacteriophage {lambda} vectors.  Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 x 107 cultured aneuploid mammalian cells (e.g., HeLa cells). - [Read Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol Protocol]

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

Protocol for isolation of T cells from synovial membranes. Includes: Initial Cell Isolation; Nonadherent Cell Isolation; Adherent Cell Isolation; Neuraminidase-Treated Sheep Red Blood Cells; Erythrocyte Lysing Solution; Balance Salt Solution (BSS). - [Read Isolation of T Cells from Synovial Membranes Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. Fluorescence for the adherent cells is measured over time with cells that have been washed free of extracellular dye. - [Read Live Single-Cell Fura-2 Measurements to Determine the Intracellular Free Calcium]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+], for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Live Single-Cell Fura-2 Measurements to Determine the Intracellular Free Calcium in RAW 264.7 Cells]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Live Single-Cell Fura-2 Measurements to Determine the Intracellular Free Calcium Protocol]

Category: Cell Culture Protocols: Cell Lysis Protocols

For cells grown in tissue culture, the most useful method of lysis is treating with detergents, as described in this protocol. Non-ionic detergents, such as NP-40, solubilize the plasma and intracellular membranes, break many weak intermolecular bonds, and solubilize most of the commonly studied protein antigens. RIPA lysis buffer may be used as a more rigorous extraction buffer to release all but the insoluble proteins of the cell and to break most weak noncovalent interactions. - [Read Lysing Tissue-Culture Cells for Immunoprecipitation Protocol]

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

This protocol describes the isolation of microsomal Endoplasmic Reticulum (ER) membranes from yeast cells. Includes: Spheroplast Formation and Microsome Purification. - [Read Microsome Preparation from Yeast Cells Protocol]

Category: Cell Biology and Cell Culture

Microtuble/ organelled motility assay using Golgi or ER membranes, 45 uM tubulin, rat liver cytosol, and 20x energy regeneration system. - [Read Microtubule/ organelle Motility Assays]