bluuebunny/crossref_metadata_2025_split huggingface dataset:
BY‐2 Cells: Culture and Transformation for Live Cell Imaging
Authors: Brandizzi Federica , Irons Sarah , Kearns Anne , Hawes Chris 
Published: June 2004

AbstractTobacco Bright Yellow‐2 (BY‐2) suspension cells are a widely used biological material for studying plant cell morphology and physiology. These cells are easy to transform and maintain in culture and tolerate transformation with fluorescent proteins such as the green fluorescent protein and its derivatives. These, by the addition of plant or mammalian targeting sequences, can be directed to specific subcellular locations for the study of cell dynamics in vivo.This unit describes the production of BY‐2 cell stable transformants via an Agrobacterium based method to permit the visualisation of cellular components in vivo by epifluorescence or confocal microscopy.
Preparation and Culture of Human Lymphocytes
Authors: Biddison William E. 
Published: June 2004

AbstractThis unit presents protocols for preparation of lymphocytes from peripheral (whole) blood or leucopherisis samples, first by differential centrifugation in Ficoll‐Hypaque to separate them from erythrocytes and granulocytes. Repeated centrifugation removes the plasma and platelets. Monocyte/macrophage cells and dendritic cells can be purified from the T and B cell population by exploiting their adhesion to serum‐coated plates in the presence of recombinant IL‐3. Different subpopulations of lymphocytes can be isolated by specific antibodies bound to magnetic beads.
Laser Capture Microdissection
Authors: Charboneau Lu , Paweletz Cloud P. , Liotta Lance A. 
Published: June 2004

AbstractThis unit describes laser capture microdissection (LCM) using the Pixcell II as a technique to provide the scientific community with the opportunity to perform molecular analyses on pure cell populations procured directly from tissues. After identifying specific cells of interest, the cells are captured by firing a near infrared laser through a thermaplastic polymer film that rests on top of the cells. The cells are then ready for molecular analyses.
Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
Authors: Tuma Pamela L. , Hubbard Ann L. 
Published: June 2004

AbstractThis unit describes a method for isolation of plasma membrane sheets from rat liver. It also includes protocols for preparation of plasma membrane domains isolated from plasma membrane sheets and indirect immunofluorescence localization of marker proteins associated with plasma membrane sheets. The unit has been updated with assays for the marker enzymes alkaline phosphodiesterase I, 5′ nucleotidase, and K+‐stimulated.
Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
Authors: Graham John M. 
Published: June 2004

AbstractDifferential centrifugation is used to prepare a “heavy” mitochondrial fraction from liver. These mitochondria are relatively pure, highly coupled, and suitable for respiratory studies. This unit presents protocols for isolation of beef heart mitochondria (also suitable for respiratory studies), skeletal muscle mitochondria, and mitochondria from cultured cells can also be isolated from homogenates by differential centrifugation. Differential centrifugation, which separates cellular organelles based on sedimentation velocity, is a rapid method for preparing mitochondria for metabolic studies.
Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient Centrifugation
Authors: Graham John M. 
Published: June 2004

AbstractPeroxisome purification depends on a two‐step procedure: differential centrifugation to prepare a light mitochondrial fraction and fractionation on a density‐gradient medium preferably iodixanol or Nycodenz, to isolate the peroxisome enriched fraction. The iodixanol gradient may be a preformed continuous gradient or a self‐generating gradient. Alternatively a continuous Nycodenz gradient or a simple Nycodenz barrier may be used for the second step. The unit contains protocols for peroxisome isolation from rat liver, tissue culture cells (HepG2 cells), and yeast spheroplasts. The extent of endoplasmic reticulum contamination of the prep can be assessed using an assay for the marker enzyme NADPH‐cytochromecreductase.
Overview of Subcellular Fractionation Procedures for the YeastSaccharomyces cerevisiae
Authors: Rieder Stephanie E. , Emr Scott D. 
Published: June 2004

AbstractThis unit provides an overview of centrifugation‐based fractionation procedures adapted for the yeastSaccharomyces cerevisiae. The goals, merits, limitations, and critical parameters of are discussed in order to facilitate the development of subcellular fractionation strategies. Topics include yeast cell lysate preparation, differential velocity centrifugation, density gradient centrifugation, and the analysis of subcellular fractions.
Isolation of Subcellular Fractions from the YeastSaccharomyces cerevisiae
Authors: Rieder Stephanie E. , Emr Scott D. 
Published: June 2004

AbstractThis unit presents detailed protocols for a range of centrifugation‐based subcellular fractionation procedures for the yeastSaccharomyces cerevisiae. Techniques include spheroplast preparation, glass‐bead lysis, differential centrifugation, and several density gradient procedures using a variety of gradient media. There are analytical procedures that are primarily designed to evaluate the association of proteins with organelles in the exocytic and endocytic pathways. Additionally, there are preparative protocols for isolation of yeast nuclei, vacuoles, mitochondria, peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. The unit also contains a table, with references, for alternative approaches to isolation of these organelles and fractions.
Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient Centrifugation
Authors: Graham John M. 
Published: June 2004

AbstractThis unit describes the isolation of Golgi membranes on a preparative basis. Methods are provided for rapid isolation of dextran‐treated Golgi stacks from rat liver using a sucrose density barrier, and Golgi isolation by floatation from a light mitochondrial fraction, along with an alternate procedure using a self‐generated iodixinol gradient. If the Golgi tends to vesiculate during homogenization (commonly the case with cultured cells), a primary requirement is to separate these vesicles from other microsomal compartments, so a procedure is described for a discontinuous gradient of sucrose for cultured cells, and an alternate protocol describes a continuous iodixinol gradient that may provide greater resolution. A self‐generated iodixinol gradient is described to prepare Golgi membranes from a microsomal fraction of rat hepatocytes, and a standard Golgi enzyme marker assay is given in a support protocol.
Isolation of Nuclei and Nuclear Membranes From Animal Tissues
Authors: Graham John M. 
Published: June 2004

AbstractThe vast majority of methods for the isolation of nuclei and nuclear membranes use soft mammalian tissues (particularly rat liver) as the biological source; however, the protocols described in this unit should be applicable generally to any tissue type, cultured cells, or cells of lower eukaryotes or plants, so long as a suitable homogenization method is available. Generally, a buffered isoosmotic medium containing KCl and MgCl2is used to stabilize the nuclei of mammalian tissues. Some of the media used for cultured animal cells and nonmammalian sources are also described. After homogenization the nuclei are purified using a sucrose barrier of 2.3 M. The use of OptiPrep overcomes the viscosity and osmolarity problems associated with the use of high molarity sucrose, and it permits the use of a high‐speed centrifuge and much shorter centrifugation times. OptiPrep is also used isolate plant cell (i.e., wheat germ) nuclei, because it is able to resolve nuclei from the denser starch granules.
Free‐Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
Authors: Fuchs Renate , Ellinger Isabella 
Published: June 2004

AbstractEndosomes constitute a functionally, morphologically, and biochemically heterogeneous population of intracellular organelles that play a major role in sorting of incoming ligands, receptors, membrane, and lumenal content. This unit provides protocols for labeling and isolation of endosomes of polarized rat hepatocytes. By application of free‐flow zone electrophoresis functional endosomal subcompartments involved in transports to lysosomes and transcytosis are resolved from each other. Methods to analyze the protein composition or acidification properties of these highly purified endosomes are also described.
Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
Authors: Brown William J. 
Published: June 2004

AbstractThis carefully written unit 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. Embedding and staining procedures are also optimized for sample visualization.
Correlative Video Light/Electron Microscopy
Authors: Polishchuk Roman S. , Mironov Alexander A. 
Published: June 2004

AbstractThis unit describes newly developed methods that allow the examination of living cells by time‐lapse analysis with the subsequent identification of the just‐observed organelle under an electron microscope. To understand how such cellular functions, such as intracellular traffic, cytokinesis, and cell migration, are organized and executed in vivo, it is most useful to observe living cells in real time with the spatial resolution afforded by electron microscopy (EM). Most suitable for this is a conceptually simple, yet powerful, method called correlative video light/electron microscopy (CVLEM), by which observations of the in vivo dynamics and the ultrastructure of intracellular objects can indeed be combined to achieve the above‐mentioned result. This unit describes this methodology, illustrates the type of questions that the CVLEM approach was designed to address, and discusses the expertise required for successful application of the technique.
Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
Authors: Waterman‐Storer Clare 
Published: June 2004

AbstractFluorescent speckle microscopy (FSM), a combination of conventional wide‐field fluorescent light microscopy and digital imaging with a low‐noise, charge‐coupled device (CCD) camera, has been developed to allow visualization of assembly/disassembly dynamics, movement, and turnover of macromolecule assemblies in vivo and in vitro. FSM uses a low level of fluorescent subunits to avoid high background. This produces an image of speckled molecules that co‐assemble with endogenous molecules and are followed to characterize dynamic events in living cells.
Total Internal Reflection Fluorescence Microscopy for High‐Resolution Imaging of Cell‐Surface Events
Authors: Jaiswal Jyoti K. , Simon Sanford M. 
Published: June 2004

AbstractThe wavelength of light imposes a physical limit of ∼400 nm on the maximum resolution that can be achieved using light microscopy. This unit will describe the use of total internal reflection fluorescence microscopy (TIR‐FM), or evanescent wave microscopy, an approach that partially overcomes this physical limit and permits one to selectively image just those fluorophores in the optical plane (along the z axis) within 50 nm of the cell surface. TIR‐FM works by means of limiting the depth of penetration of the excitation light within this narrow region. This narrow excitatory plane not only provides a high signal‐to‐noise ratio but also minimizes the photodamage to the cell.
Fluorescent Labeling of Yeast
Authors: Baggett J.J. , Shaw J.D. , Sciambi C.J. , Watson H.A. , Wendland B. 
Published: June 2004

AbstractThis unit describes the use of several different fluorescence methods for labeling yeast cells. It includes methods to label the vacuole, the actin cytoskeleton, and chitin deposits on cell walls (bud scars), as well as methods for visualizing specific proteins in live cells with GFP chimeras and in fixed cells by immunofluorescence.
Determining the Topology of an Integral Membrane Protein
Authors: Green Neil , Fang Hong , Kalies Kai‐Uwe , Canfield Victor 
Published: June 2004

AbstractA variety of methods have been developed for assigning the aqueous domains of integral membrane proteins to either side of a biological membrane. Once the sequence of a protein is known from its DNA sequence it is possible to study the topology of the protein. This unit provides protocols in which the water‐soluble domains can be tested for their accessibility to reagents added to membranes with a defined orientation. Tagging of hydrophilic regions of the protein with different epitopes and probing of their orientation with respect to the membrane is also described. Finally, a procedure for fusion of a reporter enzyme to truncated fragments of the protein is provided. The fusion protein is used as a sensor of sequence disposition relative to the membrane.
Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
Authors: Marks Michael S. 
Published: June 2004

AbstractThe molecular weight of a protein is a basic characteristic that can only be approximated by techniques such as gel filtration and electrophoresis. Zonal sedimentation analysis on sucrose gradients is a method for estimating the molecular mass of proteins and protein complexes under nondenaturing conditions. This unit includes protocols for preparing the appropriate gradients, for fractionation and separation of cell lysates on the gradients, for fractionation of the gradients themselves, and use of the results to calculate the molecular mass based on sedimentation coefficient and other parameters. There is also an additional protocol for differential sedimentation on gradients made with water and deuterium oxide to allow for direct determination of the partial specific volume of a protein or complexes.
Determination of Molecular Size by Size‐Exclusion Chromatography (Gel Filtration)
Authors: Irvine G. Brent 
Published: June 2004

AbstractSize‐exclusion or gel filtration chromatography is one of the most popular methods for determining the sizes of proteins. Proteins in solution, or other macromolecules, are applied to a column with a defined support medium. The behavior of the protein depends on its size and that of the pores in the medium. If the protein is small relative to the pore size, it will partition into the medium and emerge from the column after larger proteins. Besides a protein's size, this technique can also be used for protein purification, analysis of purity, and study of interactions between proteins. In this unit protocols are provided for size‐exclusion high‐performance liquid chromatography (SE‐HPLC) and for conventional gel filtration, including calibration of columns (in terms of the Stokes radius) using protein standards.
Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass Spectrometry
Authors: Delahunty Claire , Yates John R. 
Published: June 2004

AbstractLiquid chromatography techniques have been successfully coupled with mass spectrometers to provide a robust method for the identification of proteins in mixtures. Chromatography can be performed in‐line with the mass spectrometer and data acquisition can be directly interfaced with search algorithms for identification by correlation with databases.
One‐Dimensional SDS Gel Electrophoresis of Proteins
Authors: Gallagher Sean R. 
Published: June 2004

AbstractElectrophoresis is used to separate complex mixtures of proteins (e.g., cell lysates, subcellular fractions, column fractions, or immunoprecipitates) to investigate subunit composition and protein purity. The standard Laemmli method is used for discontinuous gel electrophoresis under denaturing conditions; it can be adapted for minigels to reduce the time requirement. Variations exist for analysis of peptides and small proteins, for analysis of proteins that require different buffer systems, for continuous gel electrophores, and for electrophoresis of different types of gels‐‐ultrathin gels, multiple single‐concentration gels, and gradient gels. Proteins in gels can be visualized by staining with protein dyes, autoradiography, or phosphorimaging.
Immunoblotting and Immunodetection
Authors: Gallagher Sean R. , Winston Scott E. , Fuller (tank transfer systems) Steven A. , Hurrell John G.R. 
Published: June 2004

AbstractThis unit provides protocols for immunoblotting, which is used to identify specific protein sequences separated by electrophoresis and transferred to an appropriate membrane and recognized by a polyclonal or monoclonal antibody. After the proteins are separated on a gel, they are transferred to a membrane by electroblotting or a semidry transfer system. Proteins on the membrane can be visualized with the reversible stain Ponceau S to assess the completeness of transfer. Then the blot is analyzed with antibodies. The primary antibody is specific for the protein(s) of interest; the secondary antibody (an anti‐Ig) is conjugated to horseradish peroxidase or alkaline phosphatase and detected colorimetrically or by chemiluminescence. The membrane can be stripped and reused for other probes.
Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
Authors: Voytas Daniel , Ke Ning 
Published: June 2004

AbstractPermanent records of the results of electrophoretic separations of radiolabeled proteins and membrane‐bound proteins (and RNA and DNA) can be made using autoradiography, fluorography, and phosphor imaging. These images can subsequently be quantified using densitometry to obtain a relative measure of the amount of radioactivity in a sample. This unit also includes descriptions of image‐enhancement techniques and guidelines for selection of appropriate recording media.
One‐Dimensional Electrophoresis Using Nondenaturing Conditions
Authors: Gallagher Sean R. 
Published: June 2004

AbstractTwo protocols for nondenaturing or native gel electrophoresis (i.e., in the absence of denaturing agents, detergent, or urea) are presented in this unit and offer a useful technique for analyzing the native size of proteins, subunit structure, and optimal separation. Mobility of proteins in native gels depends on size, shape, and intrinsic charge. Continuous PAGE is very flexible and permits cationic and anionic separations over a broad range of pH. Discontinuous PAGE is limited to separations of proteins that are negatively charged at neutral pH.
Agarose Gel Electrophoresis of Proteins
Authors: Krizek Dennis M. , Rick Margaret E. 
Published: June 2004

AbstractThis unit describes electrophoretic separation and identification of large proteins from a complex protein mixture. Agarose gel is utilized for the electrophoretic matrix, and detection of proteins is accomplished by transfer of the proteins to a membrane that is probed with specific antibodies and chemiluminescence reagents. Alternatively the protein can be detected in the gel using radiolabeled antibodies and autoradiography.
Digital Electrophoresis Analysis
Authors: Medberry Scott , Gallagher Sean 
Published: June 2004

AbstractDigital recording of gel images offers several advantages over conventional photography. Capture of the image is quick and reliable, retaining the image in a medium that allows digital analysis of the image. These images are easy to handle, accurate, reproducible and less expensive to generate. This unit provides a guide to digital capture and analysis, discussing the equipment and methods for image capture and the process of image analysis for one‐ and two‐dimensional gels.
Immunoprecipitation
Authors: Bonifacino Juan S. , Dell'Angelica Esteban C. 
Published: June 2004

AbstractSelective immunoprecipitation of proteins is a useful tool for characterizing proteins and protein‐protein interactions. Clear step‐by‐step protocols are provided for preparing lysates of cells and yeast under a variety of conditions, for binding the antibody to a solid matrix, and for performing the actual immunoprecipitation. An additional method is provided for increasing the specificity of the technique by reprecipitating the antigen with the same or a different antibody.
Metabolic Labeling and Immunoprecipitation of Yeast Proteins
Authors: Graham Todd R. 
Published: June 2004

AbstractThe proteins of Saccharomyces cervsiae can be metabolically labeled, as described here, with 35methionine and 35cysteine or a hydrolysate of E. coli labeled with 35O42−. After the labeling, protocols are provided for the mechanical disruption of the yeast cells or conversion to spheroplasts, with subsequent lysis before immunoprecipitation of the proteins.
Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
Authors: Radhakrishna Harish 
Published: June 2004

AbstractThe genetics of Drosophila is a powerful tool in the analysis of mutants and mutant proteins. Cultures of cells derived from wild‐type or mutant flies can be pulse labeled to biosynthetically label the proteins made by the cells. Immunoprecipitation (and subcellular fractionation) are used to characterize the expression of specific proteins.
Metabolic Labeling of Glycoproteins with Radioactive Sugars
Authors: Snider Martin D. 
Published: June 2004

AbstractThis unit describes methods for preparation of glycoproteins metabolically labeled with radioactive sugars, sulfate, and phosphate. Methods for liberation of both N‐ and O‐linked glycans are also described. These protocols can be used to generate materials for characterization of glycoprotein glycans from cultured cells.
Analysis of Oxidative Modification of Proteins
Authors: Yan Liang‐Jun , Sohal Rajindar S. 
Published: June 2004

AbstractReactions between protein molecules and reactive oxygen species (ROS) often lead to the modification of certain amino acid residues such as histidine, lysine, arginine, proline, and threonine, forming carbonyl derivatives. Carbonylation of proteins has thus often been employed for the quantification of generalized protein oxidation. Besides carbonylation, other types of oxidative damage that have been investigated in depth are the modifications of cysteine, tyrosine, and aspartate, or asparagine residues. Except for cysteine residues, whose oxidation is often determined by the loss of protein thiol groups, quantification of oxidative damage to tyrosine, and aspartate residues is usually carried out by the measurement of specific oxidation products such as dityrosine, nitrotyrosine (when nitrogen species are the oxidants), and isoaspartate. Methods described in this unit include spectrophotometry, immunoblotting, radiolabeling, GC/MS, ELISA adapted for analysis of oxidative modification.
Overview of the Cell Cycle
Authors: Park Michele S. , Koff Andrew 
Published: June 2004

AbstractProgression through the cell cycle is regulated by inductive signals from outside the cell and intracellular signal pathways, while the cycle itself is regulated by cyclin‐dependent kinases (CDKs). An understanding of the functions of these molecules is necessary to understand the processes of mitosis, differentiation, senescence, apoptosis, and tumorigenesis. This overview reviews the current state of knowledge for the biology of the cell‐cycle, the CDKs, the role of proteolysis, targets of the cell cycle machinery, and a paradigm of cell cycle analysis.