membrane Protocols

Category: Molecular Biology Protocols: Carbohydrate Protocols

Indirect method measuring immunofluorescence coupled to second antibody. Best for membrane antigens in addition to intra- and extracellular antigens, may be applied to frozen tissue sections, to cells in suspension, and to cells attached to glass slides or coverslips. Tadashi Tai~Head, Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan - [Read Immunohistochemistry using Anti-Ganglioside Antibodies]

Category: Immunohistochemistry Protocols

Describes two methods for using the immunoperoxidase reaction to localize antigens at the electron microscope level; one for adherent cultured cells and one for tissue sections. The reaction conditions are first optimized at the light microscope level and then adapted for EM level observation. These methods allow for reliable detection of antigens at the cell surface, within the cell, and especially in membrane bounded organelles. - [Read Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues]

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

With this protocol, transcripts that were initiated from specific genes by RNA polymerases prior to permeabilization can be measured. Instead of a nuclear extract, permeabilized cells are used. Includes information on: Permeabilization of Cells; In vitro Transcription Reaction (Run-off); Isolation of RNA; Preparation of Slot Blot Membrane for Hybridization; Hybridization of Nitrocellulose Membrane; TCA Precipitation to Determine Incorporation of [32P] GTP into Nucleic Acid - [Read In Vitro Transcription Assay (Run-off Assay) using Permeabilized Cells]

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

LCM utilizes an infrared laser integrated into a standard microscope. A transparent cap is attached to a thermoplastic transparent membrane which lies directly on the surface of a routinely prepared tissue section on a glass slide. The investigator examines the tissue section microscopically and activates the laser when the desired cells underlie the target. This in turn activates the membrane with subsequent binding and procurement of the cells of interest. - [Read Laser Capture Microdissection (LCM)]

Category: Lipid Protocols

Protocol for lipid extraction from plasma membrane fractions prepared from yeast cells. - [Read Lipid Extraction from Plasma Membrane Fractions Prepared from Yeast Cells Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol describes a method for quantitative measurement of DNA using propidium iodide (PI) staining and flow cytometry. PI stains all double-stranded regions of both DNA and RNA by intercalating between the stacked bases of the double helix. PI cannot penetrate an intact cell membrane; therefore, cells are fixed prior to staining. The ethanol-fixed cells can be stored unstained at 4°C for days, or even weeks, and then stained and analyzed. - [Read Measurement of DNA Content Using Propidium Iodide (PI) Staining of Fixed Whole Cells Protocol]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Protocol describes, samples containing the target protein are deposited onto a polyvinyldifluoride (PVDF) membrane using a vacuum manifold.  The immobilized protein is exposed to an antibody specific for the target protein, followed by an antibody that reacts with species-specific determinants carried by the primary antibody and is conjugated to horseradish peroxidase (HRP). - [Read Measuring Protein Concentration by Western Analysis Using Enhanced Chemiluminescence Detection]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

Membrane permeability of perfused cell cultures, as determined by the efflux of [3H]-2-deoxy-D-glucose-6phosphate, is used as an indicator of the cytotoxic effect of chemicals. - [Read Membrane Permability as a Measure of Cytotoxcity in Perfused Cell Cultures]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol for membrane potential analysis by flow cytometry. Includes: Preparation of the dyes; Loading of the dyes; Flow Cytometric Analysis. - [Read Membrane Potential Analysis by Flow Cytometry Protoocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Several common drugs, their targets, and protocols are described for studying organelle distribution and trafficking. The drugs are readily available from general suppliers, including Sigma, Roche, and Calbiochem. - [Read Membrane Trafficking and Organelle Reagents]

Category: RNA Protocols: cDNA Libraries Library

Purpose: To transfer DNA from bacterial colonies to nylon (or nitrocellulose) membrane so that a library can be screened by hybridization. Jim Howe Donis Keller Lab - [Read Method: Transfer of DNA from Bacterial Colonies to Nylon Membrane or Nitrocellulose]

Category: Cell Organelle Protocols: Mitochondria Protocols

Protocol describes a method for the evaluation of mitochondrial function using the fluorochrome CMXRos. CMXRos is sequestered by actively respiring mitochondria, but washed out when the mitochondrial membrane potential is lost. This analysis can be combined with the TUNEL technique or immunocytochemistry. - [Read Microscopic Analysis of Mitochondrial Transmembrane Potential Protocol]

Category: Cell Organelle Protocols: Mitochondria Protocols

Kit for detecting the mitochondrial membrane potential. - [Read Mitochondrial Membrane Potential Detection Kit]

Category: Neuroscience: Neuron Cell Culture Protocols

Neuron Cell Culture Solutions, Neuron Culturing, Preparation of Brain Membrane Fractions, Biotinylation Assay of Receptor Endocytosis, PSD Prep. Ehlers lab Duke Univ. - [Read Neuron Culture Solutions]

Category: RNA Protocols: cDNA Libraries Library

Denaturation Solution, Neutralization Solution, Rinse Solution, Crosslink DNA to membranes using Stratalinker UV crosslinker. Donelson Lab, Univ. Iowa. - [Read Nitrocellulose Membrane Washing Procedure for cDNA Screening]

Category: Fungi Protocols: Neurospora Protocols

Northern blot protocol for the detection of RNA in Neurospora. Includes: Extract RNA from tissue powder; Electropheresis and transfer RNA; Preparing the membrane for probing and preparation of a riboprob.; Hybridization. - [Read Northern blot Protocol for the Detection of RNA in Neurospora]

Category: Histology Protocols: Tissue Sample Sectioning

Generally Required Materials and Equipment for Tissue Sectioning. Sectioning and Slide Preparation Procedure onto Metal-Framed PEN Membrane Slides. Arcturus. - [Read Optimized Protocol for Mounting Tissue Sections PDF]

Category: Transfection Protocols

Protocol describes transfection of plasmid DNA into mammalian cell lines using electroporation, a process whereby external application of electric pulses induce cell membrane permeability. Cells in suspension and small volume cells are difficult to transfect, whereas adherent cells and large volume cells are relatively easy. Regardless of cell size or phenotype, transfection efficiency increases with a high concentration of cells in a small volume. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol describes transfection of plasmid DNA into mammalian cell lines using electroporation, a process whereby external application of electric pulses induce cell membrane permeability. A number of factors can affect electrotransfection efficiency. In general, cells in suspension and small volume cells are difficult to transfect, whereas adherent cells and large volume cells are relatively easy. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Paraformaldehyde Fixation of Cells protocol. This fixation method is good for cells labeled by fluorochrome-conjugated antibodies to membrane antigens.  It will stabilize the light scatter and labeling for up to a week in most instances, allowing you to be more flexible in scheduling cytometer time.  Furthermore, it inactivates most biohazardous agents, so it is important from a safety standpoint as well. Iowa University. - [Read Paraformaldehyde Fixation of Cells]

Category: Protein Protocols: Western Blot Protocol

Western blotting using enriched membrane preparation and postsynaptic densities are desired. (BD Biosciences) - [Read Preparation of Brain Membrane Fractions for Western Blot Analysis]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. - [Read Preparation of Cells and Reagents for Flow Cytometry Protocols]

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: Fungi Protocols: Neurospora Protocols

Protocol for Preparing Vacuolar Membranes And Other Membrane Fractions from Neurospora Crassa (4 liter prep, easily scaled up to 10, 12, 24, or 30 l). - [Read Preparing Vacuolar Membranes And Other Membrane Fractions from Neurospora Crassa Protocol]

Category: Cell Biology and Cell Culture

Matirgel is considered as basement membrane and generated from EHS sarcoma. Matrigel contains not only basement membrane components (collagens, laminin, and proteoglycans)but also matrix degrading enzymes/their inhibitors and growth factors. Invasion of tumor cells into Matrigel has been used to characterize involvement of ECM receptors and matrix degrading enzymes which play roles in tumor progression. - [Read Protocol for Matrigel Invasion Assays]