resolution 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: Drosophila Protocols

Protocol for antibody addition to Drosophila specimens and detection using fluorochrome-linked reagents. Fluorochrome-linked reagents should be used when high resolution is needed or if two antigens need to be localized simultaneously. Because of the thickness of fly specimens, detection requires access to a confocal microscope. - [Read Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Manual measurement and manipulation of the cell surface requires access to the cells, usually in an open chamber. Temperature-controlled chambers or stage inserts are preferred for maintaining physiological activity during the experiment. For example, heated culture dishes with coverslip glass bottoms (Bioptechs) permit high-resolution fluorescence microscopy of living cells during force application. - [Read Chambers for Examination of Live Cells under Mechanical Stress Protocol]

Category: Microarray Protocols: Microarray Chip Protocols

Dnase-chip: A High Resolution Method to Identify DnaseI Hypersensitive Sites using Tiled Microarrays. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome. - [Read Dnase-chip Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Flow cytometric analysis of gram-negative bacterial cells. Includes: Staining Conditions and FACSort Settings; Light Scatter Resolution on the BD FACSort; Bacterial Cell Resolution on the BD FACSort. - [Read Flow Cytometric Analysis of Gram-Negative Bacterial Cells]

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: Cell Culture Protocols

For low-resolution work, cells to be used for staining can be grown directly on regular tissue-culture dishes. It is a convenient method that does not require much preparatory work. - [Read Growing Adherent Cells Directly on Tissue Dishes Protocol]

Category: Cell Culture Protocols

Glass is an excellent substrate for most tissue-culture-adapted cells and is compatible with all fixing and staining solutions. Glass coverslips in tissue-culture dishes or in 24-well multiwell plates are suitable carriers, as are multiwell slides. For high-resolution studies, choose glass coverslips of the highest available grade; #1 or #1.5 coverslips are the appropriate thickness. - [Read Growing Adherent Cells on Coverslips or Multiwell Slides Protocol]

Category: DNA Protocols: SNP Protocols

High-resolution SNP mapping by denaturing HPLC. A SNP mapping procedure that relies on resolving polymorphisms by denaturing HPLC without the necessity of determining the nature of the SNPs. They demonstrate the use of denaturing high-performance liquid chromatography to identify mutations in the candidate genes and to fine-map chromosomal breakpoints. - [Read High-resolution SNP mapping by denaturing HPLC]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

The same GFP-tagged actin construct used in cell transfection experiments has been used to produce transgenic mice. Transgenic animals allow the imaging of brain tissue in the intact animal, as acutely cut slices or as organotypic slice cultures. They also serve as a source of cells for imaging neurons at high resolution in dispersed low-density cell culture. In contrast to cells transfected in culture, where the level of actin-GFP expression in neurons varies considerably, transgenic mice... - [Read Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol]

Category: Microscopy Protocols

The atomic force microscope (AFM) is one of the most powerful tools for determining the surface topography of native biomolecules at subnanometer resolution. The AFM can also provide insight into the binding properties of biological systems. In order to determine the specific interaction between two kinds of molecules (e.g., avidin and biotin). Includes information on principle of AFM and application of AFM. - [Read Imaging, Measuring and Manipulating Native Biomolecular Systems with the Atomic Force Microscope]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Live nematodes can be studied at all levels of microscopic resolution. Data collection includes information on: Photographic Records, Video Microscopy,Multiple-Focal-Plane Time-Lapse Recording Systems and Digital Imaging. - [Read Live Imaging of Caenorhabditis elegans: Observation of Nematodes and Data Collection]

Category: C. elegans Protocols

At low magnification, dissection microscopes are useful, whereas at higher magnification and resolution, a wide range of optical methods can be used, including differential interference contrast (DIC) optics, phase contrast, fluorescence, polarized light, confocal, and dark field. - [Read Live Imaging of Caenorhabditis elegans: Observation of Nematodes and Data Collection Protocol]

Category: Microscopy Protocols: Optical Microscopy Protocols

Diffraction-limited optical microscopy requires that the spatial resolution of an image is limited by the wavelength of the incident light & by the numerical apertures of the condenser & objective lens systems.The development of near-field scanning optical microscopy (scanning near-field optical microscopy) has allowed for a imaging technique that retains the various contrast mechanisms afforded by optical microscopy methods while attaining spatial resolution beyond the optical diffraction limit - [Read Near-Field Scanning Optical Microscopy]

Category: Microscopy Protocols: Optical Microscopy Protocols

Near-field scanning optical microscopy can achieve spatial resolution performance beyond the classical diffraction limit by employing a sub-wavelength light source or detector positioned in close proximity to a specimen. Such a sub-wavelength source usually consists of an aperture at the end of a tapered probe, which functions basically as a wave guide. Includes info.: Fiber Probe Fabrication; Pulling Method; Meniscus Etching; Selective Etching; Apertureless and Alternative Probe Designs etc. - [Read Near-Field Scanning Optical Microscopy: NSOM Probes]

Category: PCR Protocols: In-situ PCR Protocols

PCR IN SITU: NEW TECHNOLOGIES WITH SINGLE CELL RESOLUTION FOR THE DETECTION AND INVESTIGATION OF VIRAL LATENCY AND PERSISTENCE. Staskus 1998 U.Minn. - [Read PCR IN SITU: NEW TECHNOLOGIES WITH SINGLE CELL RESOLUTION]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the preparation of ion-exchange chromatography column.  Ion-exchange chromatography (IEC) can be used as a crude step in a protein purification scheme, or, with proper preparation, as a high-resolution step.  If high resolution is desired, considerable care should be taken during column preparation, choice of IEC media, and column packing. - [Read Preparation of an Ion-Exchange Column Protocol]

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: Immunology

Removal of CCR5 ligands and induction of pro-resolving lipid mediators by apoptotic neutrophils during resolution. Application of lipid extraction from peritoneal exudates, in tandem with lipid mediator informatics can be used to determine the role of apoptotic neutrophils in the generation of resolution phase lipid mediators. This neutrophil transfer system allows the determination of the direct impact of apoptotic leukocytes in the resolution of inflammation. - [Read Removal of CCR5 Ligands and Induction of Pro-Resolving Lipid Mediators by Apoptotic Neutrophils]

Category: Immunohistochemistry Protocols: Resin Staining Protocols

Protocol for resin embedding for high resolution morphology. - [Read Resin Embedding for High Resolution Morphology Protocol]

Category: Nucleic Acid Detection Protocols

Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment.  It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome.  RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. - [Read Restriction Landmark Genomic Scanning Protocol]

Category: DNA Protein Interactions Protocols: Electrophoretic Mobility Shift Assays EMSA Protocols

The target sequence is simultaneously labeled and amplified, then heat-denatured and resolved by non-denaturing polyacrylamide gel electrophoresis.  Differences in sequence alter the conformation of the DNA and hence its electrophoretic mobility and, because of the high resolution of polyacrylamide gels, most conformational changes caused by subtle changes in sequence can be detected. - [Read Single-Strand Conformation Polymorphism Analysis Protocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Sophisticated fluorescence microscopy methods & equipment, now allow cellular events to be studied at high resolution in living material. The studying of living fly tissues presents unique difficulties in keeping the cells alive, introducing fluorescent probes, & imaging through thick hazy cytoplasm. This protocol outlines the preparation of major tissue types amenable to study by time-lapse cinematography and different methods for keeping them alive. - [Read Time-Lapse Cinematography in Living Drosophila Tissues: Preparation of Material]

Category: Protein Protocols: Protein Electrophoresis

Tricine–SDS-PAGE Protocol and background. Nature. PDF file. Tricine–SDS-PAGE is commonly used to separate proteins in the mass range 1–100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. –SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification. - [Read Tricine–SDS-PAGE Protocol PDF]

Category: Microscopy Protocols: Optical Microscopy Protocols

The light microscope allows dynamic biological processes to be imaged in their native (i.e., aqueous) environment with relatively high temporal resolution. However, the diffraction-limited resolution is low. When working at or beyond the diffraction-limited resolution of the LM, a disadvantage of fluorescence imaging is the relatively low signal-to-noise (S/N) ratio of the images. However, this can be increased significantly by video and computer technology. - [Read Watching Molecular Motors at Work by Video-Enhanced Light Microscopy]