bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of Nuclei and Nuclear Membranes From Animal Tissues
Authors: Graham John M.
Published: June 2004

The 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

Endosomes 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.
Basic Confocal Microscopy
Authors: Smith Carolyn L.
Published: June 2004

This comprehensive overview unit introduces the reader to confocal microscopy from the basic principles of imaging and optical sectioning, to selection of laser, practical guidelines for fixation, choice of fluorophore, control samples, and mounting the sample. There are also suggestions for optimizing the imaging parameters.
Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
Authors: Brown William J.
Published: June 2004

This 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

This 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

Fluorescent 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

The 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

This 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

A 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

The 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

Size‐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

Liquid 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

Electrophoresis 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

This 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

Permanent 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

Two 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

This 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

Digital 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

Selective 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

The 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

The 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

This 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

Reactions 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

Progression 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.
Assays for CDK Activity and DNA Replication in the Cell Cycle
Authors: Pines Jonathon , Jackman Mark , Simpson Karen
Published: June 2004

This unit describes two assays for different stages of the cell cycle in tissue culture cells. One is a biochemical measurement that assays the protein kinase activity of different cyclin‐dependent kinase complexes that are present in late G1 phase, S phase, G2 phase, or mitosis. The other assay uses immunofluorescence to detect DNA replication by the incorporation of nucleotide analogs into DNA. These assays are useful in analyzing the stage and degree of synchrony of the cell cycle and changes in the basic cell cycle machinery.
Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
Authors: Jackman Joany , O'Connor Patrick M.
Published: June 2004

Exponentially growing cells are asynchronous with respect to the cell cycle stage. Detection of cell cycle‐related events is improved by enriching the culture for cells at the stage during which the particular event occurs. Methods for synchronizing cells are provided here, including those based on morphological features of the cell (mitotic shake‐off), cellular metabolism (thymidine inhibition, isoleucine depravation), and chemical inhibitors of cell progression in G1 (lovastatin), S (aphidicolin, mimosine), and G2/M (nocodazole). Applications of these methods and the advantages and disadvantages of each are described.
Determining Cell Cycle Stages by Flow Cytometry
Authors: Darzynkiewicz Zbigniew , Juan Gloria , Bedner Elzbieta
Published: June 2004

This unit describes assays used to determine the distribution of a population of cells to the different stages of the cell cycle as analyzed by flow cytometry. Staining the DNA with different fluorescent dyes, propidium iodide or DAPI, is one of the most direct ways of staging the cells based on DNA content. These staining procedures can be used with fixed cells or detergent‐permeabilized cells. Alternatively, live cells can be stained with the membrane‐permeable stain, Hoechst 33242, and sorted on the basis of DNA content for further culture and characterization. When DNA content measurements are coupled with immunostaining for cyclin expression, it is possible to reliably detect even more stages in progression through the cell cycle.
Cadherin‐Dependent Cell‐Cell Adhesion
Authors: Takeichi Masatoshi , Nakagawa Shinichi
Published: June 2004

Differential treatment of cells with trypsin can be used to distinguish Ca2+‐dependent adhesion (CDS) from Ca2+‐independent adhesion (CIDS). Cadherins appear to be a unique family of molecules whose structure and function as adhesion molecules
are protected from trypsin in the presence of Ca2+. This unit
provides protocols for preparation and analysis of cells for
cadherin‐dependent adhesion in short‐term and long‐term
aggregation assays. The functions of different cadherins can be assessed in
mixed aggregate assays. Fluorescence antibody‐based assays are used to
identify specific cadherins and their associated catenins, and transformation
of cells with specific constructs can be used to assay adhesion in cells with
loss of cadherin activity.
Analysis of Cell‐Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
Authors: Sonderegger Peter , Kunz Stefan , Rader Christoph , Suter Daniel M. , Stoeckli Esther T.
Published: June 2004

This unit provides protocols to assay cell‐cell adhesion and adhesive‐dependent cellular functions mediated by calcium‐independent adhesion molecules. These protocols have been developed for neural cell adhesion molecules of the Ig superfamily. However, most of the protocols allow a more general application to other categories of adhesion molecules and non‐neural cells.
Overview of Extracellular Matrix
Authors: Mecham Robert P.
Published: June 2004

The extracellular matrix provides an environment for cells. It is produced, assembled and modified by cells and in turn, it modifies the functions and behavior of the cells it encounters. The molecules that make up the matrix are diverse in both structure and function. This well‐illustrated unit provides an introduction to the structure and function of the major components of matrix and serves as a background for the other units in the chapter which include protocols for isolation and analysis of individual components.
Preparation of Basement Membrane Components from EHS Tumors
Authors: Kleinman Hynda K.
Published: June 2004

This unit describes methods for passaging and harvesting the basement membrane matrix‐producing EHS tumor and for the subsequent isolation of a crude mixture of basement membrane components termed Matrigel, which promotes the differentiation of a variety of epithelial, endothelial, and neuronal cells. Procedures for the isolation of the adhesive glycoprotein laminin‐1 and of type IV collagen are also included. Support protocols cover the maintenance and harvesting of EHS tumors in mice and maintenance of mice on a lathrogenic diet.
Preparation of Gelled Substrates
Authors: Kleinman Hynda K.
Published: June 2004

Gelled substrates can be used for a variety of in vitro and in vivo experiments. A type I collagen gelled substrate will promote cell growth and differentiation. Gelled Matrigel substrate promotes the survival of explanted cells and tissues and the differentiation of a variety of epithelial and endothelial cell types in vitro and to assess angiogenesis and increase tumor growth in vivo. Preparation of the matrices and their use are described in this unit.
Purification of Fibronectin
Authors: Akiyama Steven K.
Published: June 2004

This unit describes the purification of the multifunctional adhesive glycoprotein fibronectin from plasma or of cell‐derived fibronectin from cell surfaces and from conditioned medium. Fibronectin can be used in cell adhesion and migration assays, and can be obtained in relatively high purity using simple affinity chromatography techniques.
Preparation of Extracellular Matrices Produced by Cultured Fibroblasts
Authors: Cukierman Edna
Published: June 2004

Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell‐to‐matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer, and other diseases. This unit describes a method for preparing a three‐dimensional matrix derived from NIH‐3T3 cells; the matrix is three‐dimensions, cell and debris free, and attached to a two‐dimensional culture surface. Cell adhesion and spreading are normal on these matrices. This matrix can also be compressed into a two‐dimensional matrix and solubilized to study the matrix biochemically.
Purification ofSPARC/Osteonectin
Authors: Sage E. Helene
Published: June 2004

SPARC is a matricellular protein that regulates cell adhesion, extracelluolar matrix production, growth factor activity and cell cycle. This unit describes the purification of SPARC, also termed osteonectin and BM/40, from cultured mammalian cells. Additional information is presented on the purification of recombinant SPARC (rSPARC) fromE. coliand from Sf9 cells, as well as its isolation from blood platelets. Assays for the activity of SPARC, de‐adhesion and inhibition of cellular proliferation in vitro, are described. The expression of SPARC during remodeling and repair tissue in response to injury identifies it as a therapeutic target for the treatment of fibrotic disease, certain cancers and other disorders in which regulation of angiogenesis is a key factor.
Overview of Eukaryotic In Vitro Translation and Expression Systems
Authors: Jagus Rosemary , Beckler Gregory S.
Published: June 2004

The ability to investigate cellular processes in vitro permits detailed analysis of the process and its molecular components. Eukaryotic translation and expression is one system that has been well studied. This overview describes the development of in vitro systems, including such approaches as continuous‐flow systems, coupled transcription/translation, and the incorporation of non‐natural amino acids. It also discusses molecular and genetic studies to probe translation, including post‐translational fate of the synthesized proteins.
In Vitro Translation
Authors: Jagus Rosemary , Joshi Bhavesh , Miyamoto Suzzane , Beckler Gregory S.
Published: June 2004

The two most frequently used systems for in vitro translation are the rabbit reticulocyte system and the wheat germ extract. These systems are useful for mRNAs isolated from cells, tissues, and capped or uncapped mRNA produced in vitro by transcription of cDNA. In a combined system, mRNA can be transcribed and translated in a single reaction. In addition these systems can be used for translation reactions with biotinylated amino acids; this allows capture of the newly synthesized protein using streptavidin immobilized on agarose.
Cotranslational Translocation of Proteins into Canine Rough Microsomes
Authors: Bernstein Harris D.
Published: June 2004

Rough microsomes (RMs) are small vesicular fragments of the rough endoplasmic reticulum (ER). They can be used in cell‐free protein translocation studies to analyze ER targeting signals, signal sequence cleavage, N‐linked glycosylation, and membrane protein biogenesis. This unit describes preparation and use of RMs.Rough microsomes (RMs) are small vesicular fragments of the rough endoplasmic reticulum (ER). They can be used in cell‐free protein translocation studies to analyze ER targeting signals, signal sequence cleavage, N‐linked glycosylation, and membrane protein biogenesis. This unit describes preparation and use of RMs.
Nuclear Import in Digitonin‐Permeabilized Cells
Authors: Moore Mary Shannon , Schwoebel Eric D.
Published: June 2004

Import of proteins into the nucleus of a cell is a complex process that can be reconstituted in vitro. Digitonin‐permeabilized cells are washed free of cytosolic factors to provide competent nuclei. Cytosolic factors for import are provided by an extract of Xenopus ovarian cells. Fluorochrome‐conjugated probes, cloned proteins fused to green fluorescent protein, or antibodies to the protein of interest are used to visualize nuclear import. The system can also be used to identify nuclear localization sequences (NLS) of imported proteins.
In Vitro Translation Using HeLa Extract
Authors: Witherell Gary
Published: June 2004

A HeLa cell extract can be used as an alternative to rabbit reticulocyte lysate (RRL) for in vitro translation of mRNAs. It is particularly suited for synthesis of proteins that cannot be translated by RRL, is more cap dependent, has a higher capacity for faithful initiation at the correct start codon, and is more likely representative of translation in intact mammalian cells. This unit provides a detailed protocol for production and use of an mRNA‐dependent cell‐free translation system from HeLa extract. A support protocol describes production and purification of the mRNA.
Analysis of Eukaryotic Translation in Purified and Semipurified Systems
Authors: Merrick William C. , Hensold Jack O.
Published: June 2004

Much of the current understanding of the sequential steps involved in translation initiation has been obtained using sucrose gradients to isolate ribosomes and ribosomal subunits, as described here. These purified components are combined with purified translation factors to analyze the formation of intermediates in translation initiation and the roles of the translation factors in vitro.
Preparation and Use of Interphase Xenopus Egg Extracts
Authors: Powers Maureen , Evans Erica K. , Yang Jing , Kornbluth Sally
Published: June 2004

In this unit, Xenopus eggs are isolated from hormonally primed female frogs, and then the extract is treated with cyclohexamide so it remains in interphase of the cell cycle. In the presence of sperm chromatin and ATP, membrane vesicles in the extract fuse to assemble nuclei, making the extract suitable for studies of DNA replication and nuclear transport.
Analysis of the Cell Cycle Using Xenopus Egg Extracts
Authors: Kornbluth Sally , Yang Jing , Powers Maureen
Published: June 2004

In this unit, Xenopus eggs are isolated from hormonally primed female frogs, and then the extract is treated with cyclohexamide so it remains in interphase of the cell cycle. In the presence of sperm chromatin and ATP, membrane vesicles in the extract fuse to assemble nuclei, making the extract suitable for studies of DNA replication and nuclear transport.
Analysis of Apoptosis Using Xenopus Egg Extracts
Authors: Kornbluth Sally , Evans Erica K.
Published: June 2004

In the presence of a subcellular fraction enriched for mitochondria, after prolonged incubation the Xenopus egg extract can mimic biochemical aspects of apoptosis such as caspase activation, and DNA fragmentation. This unit describes preparation of an apoptotic extract from a crude interphase extract and an extract fractionated into latent and execution phases. An apoptotic extract can also be reconstituted from a fractionated interphase extract and purified mitochondria. Protocols are also included for monitoring apoptotic progression in the extract either by following activation of apoptotic proteases (caspases) or by assessing translocation of cytochrome c from the mitochondria to the cytosol.
Mitotic Spindle Assembly In Vitro
Authors: Merlie John , Heald Rebecca
Published: June 2004

The protocols in this unit describe the preparation of materials for an in vitro assay of mitotic spindle assembly in Xenopus egg extracts. Fluorochrome‐labeled tubulin is used to visualize microtubule asters and spindles.
In Vitro Analysis of Peroxisomal Protein Import
Authors: Terlecky Stanley R.
Published: June 2004

This unit describes a quantitative in vitro assay for peroxisomal protein import that accurately reconstitutes established properties of the pathway. The cell‐free system is ELISA based and employs semi‐permeabilized human cells with a biotinylated peroxisomal targeting signal‐containing substrate.
Chemotaxis Assays for Eukaryotic Cells
Authors: Zigmond Sally H. , Foxman Ellen F. , Segall Jeffrey E.
Published: June 2004

Chemotaxis is a complex response of a cell to an external stimulus. It involves detecting and measuring the concentration of the chemoattractant, biochemical transmission of the information, and the motility and adhesive changes associated with the response. This unit describes a number of chemotaxis assays that can be used to identify chemoattractants individually and in large‐scale screenings, to distinguish chemotaxis from chemokinesis, and to analyze cellular behavioral and biochemical responses. Some of these assays such as the filter, under agarose, and small population assays, can be used to monitor the behavior of large groups of cells; the bridge, pipet, and upshift assays can be used to analyze the responses of single cells.
Invasion Assays
Authors: Kleinman Hynda K. , Jacob Karin
Published: June 2004

Basement membrane is a thin extracellular matrix that underlies epithelia and endothelia and separates them from the stroma. Tumor cells must cross this membrane to invade stroma and establish distant metastases. They do this by producing proteases that degrade the matrix. Of several in vitro models, those using Matrigel are the most reliable, reproducible, and representative of in vivo invasion. In the assay presented here, tumor cells are placed in the upper chamber of Boyden migration chamber. The upper and lower chambers are separated by a porous membrane coated with Matrigel. A chemoattractant in the lower well stimulates migration. After an interval, tumor cells are recovered from or counted on the lower surface of the membrane.
Dictyostelium Cell Dynamics
Authors: Parent Carole A.
Published: June 2004

This unit describes culturing and imaging of D. discoideum amoebae to study fundamental cellular responses, such as motility and directed migration. The system displays powerful molecular genetics that can be used to link structural determinants of proteins with in vivo cellular functions.
Microtubule/Organelle Motility Assays
Authors: Waterman‐Storer Clare M.
Published: June 2004

This unit describes an in vitro assay that uses video‐enhanced differential interference contrast (VE‐DIC) microscopy to examine the motile interactions between isolated organelle fractions and microtubules (MTs). The method can be used to dissect the molecular requirements for organelle movement and membrane trafficking. A field of axoneme‐nucleated MTs, growing and shortening as they would in a living cell (dynamic MTs), is generated in a simple microscope perfusion chamber. Various combinations of isolated endoplasmic reticulum (ER) and Golgi apparatus organelles, cytosol containing motor proteins and other soluble factors, nucleotides, and specific pharmacological reagents are then added to the dynamic MT, and the motile interactions between the organelles and MTs are observed by VE‐DIC microscopy.
Movement of Nuclei
Authors: Reinsch Sigrid
Published: June 2004

This unit describes the first assay that reconstructs the movement of the female pronucleus in the newly fertilized frog egg. Nuclei are assembled in frog egg extracts and translocated along microtubules using the microtubule motor dynein.
Measuring Dynamics of Nuclear Proteins by Photobleaching
Authors: Dundr Miroslav , Misteli Tom
Published: June 2004

The mobility of nuclear proteins can be studied by photobleaching techniques. The three main advantages of photobleaching are fast experimental turn around, good spatial and temporal resolution, and the ability to measure kinetics inside of living cells. The main disadvantage of these techniques is the requirement for fluorescently tagged proteins that have rigorously tested to ensure it has the same properties and function as its native counterpart. Three major methods of photobleaching microscopy are described: fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and inverse fluorescence recovery after photobleaching (iFRAP). Each of these techniques has characteristics permitting the determination of distinct parameters of protein behavior in vivo.
Overview of Protein Phosphorylation
Authors: Sefton Bartholomew M.
Published: June 2004

Phosphorylation is the most common and important mechanism of acute and reversible regulation of protein function. Studies of mammalian cells metabolically labeled with [32P]orthophosphate suggest that as many as one‐third of all cellular proteins are covalently modified by protein phosphorylation. Protein phosphorylation has an important role in essentially all aspects of cell biology. Most polypeptide growth factors (platelet‐derived growth factor and epidermal growth factor are among the best studied) and cytokines (e.g., interleukin 2, colony stimulating factor 1, and γ‐interferon) stimulate phosphorylation upon binding to their receptors. Induced phosphorylation in turn activates cytoplasmic protein kinases, such as Raf, the activators of the mitogen‐activated protein (MAP) kinases SEK and MEK, the MAP kinases ERK, JNK, and p38, the Janus/JAK kinases, the p21 activated kinases (PAKs), and the phosphatidylinsoitil 3'‐kinase‐activated kinase, protein kinase B/Akt. Additionally, in all nucleated organisms, cell cycle progression is regulated at both the G1/S and the G2/M transitions by cyclin‐dependent protein kinases. These kinases regulate the G1/S transition by the phosphorylation of cell cycle regulators such as Rb protein and the G2/M transition through the phosphorylation of nuclear lamins and histones.
Immunological Detection of Phosphorylation
Authors: Yao Zhong , Seger Rony
Published: June 2004

Incorporation of phosphates into serine, threonine, and tyrosine acceptors in proteins is a common mechanism for regulating protein function. This unit presents protocols that use specific anti‐phosphoamino acid (PAA) and anti‐phosphoprotein antibodies to detect protein phosphorylation and protein kinase activity. Immunoblotting to detect protein phosphorylation using either anti‐PAA or anti‐phosphoprotein antibodies. This is a convenient method that usually yields impressive results. Phosphorylation can also be detected by immunoprecipitation followed by immunoblot analysis or by immunofluorescent staining; these methods are typically more complicated and time consuming. All three methods have been successfully used to detect protein phosphorylation with a wide variety of antibodies and most phosphorylated proteins.
Detection of MAP Kinase Signaling
Authors: Rubinfeld Hadara , Seger Rony
Published: June 2004

Elucidation of intracellular signaling pathway is essential to understanding the regulatory mechanisms of cellular function. Mitogen‐activated protein kinase (MAPK) signaling cascades are a major pathway in intracellular signaling. The methods used to detect cascade activation involve measurements of protein kinase activities. This unit describes an assay for specific immunoprecipitation of a protein kinase and detection of phosphorylation of the appropriate substrate, an in‐gel kinase assay for kinases of unknown identity or for which there are no specific antibodies, and the JNK assay, which involves a broad‐based immunoaffinity purification followed by phosphorylation.
Phosphoamino Acid Analysis
Authors: Sefton Bartholomew.M.
Published: June 2004

Proteins involved in signal transduction are often phosphorylated. Determination of the specific amino acid residue(s) involved is used in characterizing the particular pathway. Partial acid hydrolysis of phosphorylated proteins followed by two‐dimensional thin layer chromatography is used to identify the phosphorylated residues of the protein as phosphoserine, phosphothreonine, or phosphotyrosine. Mild alkaline hydrolysis is used to enhance detection of phosphothreonine and phosphotyrosine.
Determination of Akt/PKB Signaling
Authors: Pankov Roumen
Published: June 2004

This unit provides a basic protocol for assaying the level of activity, as well as the activation capacity and dynamics of inhibition of Akt/PKB in cultured cells. All these data can be obtained in a single nonradioactive experiment by standard techniques including immunoblotting and immunodetection with phosphospecific antibodies.

This unit also provides a support protocol for assaying the membrane translocation of Akt.
Determining Protein Transport to the Plasma Membrane
Authors: Neefjes Jacques
Published: June 2004

Many integral membrane proteins synthesized in the endoplasmic reticulum ultimately arrive at the cell surface to contact the cell environment. During transit through the Golgi and trans‐Golgi network, proteins acquire post‐translational modifications that can be used to track the appearance of such modified proteins at the cell surface. Cellular proteins can be treated with enzymes–e.g., sialidase or protease–or antibodies, or biotinylated to identify molecules that have reached the cell surface. Some proteins first enter the endocytic pathway before appearing at the cell surface; this is detected by treating the cells at 4o and 37oC. Analysis of the number of sialic acids on proteins of cells treated at 4oC identifies proteins resident at the cell surface, while cells treated at 37oC internalize the sialidase, which can then act with proteins in the endocytic compartments.
Analysis of Membrane Traffic in Polarized	Epithelial Cells
Authors: Lipschutz Joshua H. , O'Brien Lucy Erin , Altschuler Yoram , Avrahami Dana , Nguyen Yen , Tang Kitty , Mostov Keith E.
Published: June 2004

Spatial asymmetry is fundamental to the structure and function of most eukaryotic cells. A basic aspect of this polarity is that the cell's plasma membrane is divided into discrete domains. The best studied and simplest example of this occurs in epithelial cells, which line exposed body surfaces. Epithelial cells use two pathways to send proteins to the cell surface. Newly made proteins can travel directly from the trans‐Golgi network (TGN) to either the apical or basolateral surface. Alternatively, proteins can be sent to the basolateral surface and then endocytosed and transcytosed to the apical surface. Epithelial cells grown on porous filters adopt a typical polarized morphology; transfected epithelial cells can be used to biosynthetically characterize the trafficking patterns of a given protein. These cells can also be used to study delivery to a particular surface and to localize the protein by immunofluorescence.
Measurements of Phagocytosis and Phagosomal Maturation
Authors: Chow Chung‐Wai , Downey Gregory P. , Grinstein Sergio
Published: June 2004

Phagocytosis is an essential component of the innate immune response. Invading microorganisms are engulfed by neutrophils and macrophages, and subsequently killed by a complex series of reactions that require fusion of the phagocytic vacuole with multiple endomembrane organelles. This vacuolar remodeling process, known as phagosomal maturation, is often the target of intracellular parasites that evade killing by the host immune system. This unit describes detailed protocols for the assessment of phagosome formation and maturation. Particular attention is given to Fc‐ and complement‐receptor mediated phagocytosis, but the protocols provided can be readily adapted to other types of phagocytic processes.
Production of Monoclonal Antibodies
Authors: Yokoyama Wayne M.
Published: June 2004

Monoclonal antibodies are particularly specific for a single epitope and can be produced in large quantities. When immune splenic cells are fused with a tumor cell line, they produce hybridomas each secreting one highly specific antibody. These cells are then characterized and cloned to allow production over time. This unit describes the production of monoclonal antibodies beginning with basic protocols for immunization and cell fusion and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium (thymocyte‐conditioned medium).
Production of Polyclonal Antisera
Authors: Cooper Helen M. , Paterson Yvonne
Published: June 2004

Polyclonal antibody preparations contain multiple antibody molecules recognizing different epitopes on an antigen molecule. Good quality polyclonal antisera are useful for a number of applications–e.g., immunoprecipitation, immunoblotting, and ELISAs–where the binding of more than one antibody molecule per antigen molecule improves the sensitivity of the assay. It is possible to produce polyclonal antibodies in a variety of species, and this make it possible to detect multiple immunoreactions. The protocols in this unit describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters using complete Freund's adjuvant (CFA) or other adjuvants. In addition, the unit presents a method for preparing serum from blood. Polyclonal antipeptide antisera can be produced by substituting carrier‐conjugated peptides for the purified protein antigens.
Purification of Immunoglobulin G
Authors: Andrew Sarah M. , Titus Julie A.
Published: June 2004

While unpurified antibodies are suitable for a number of applications, purified antibodies are required for assays based on known concentration of antibody, for chemical modifications such as radiolabeling or conjugation with fluorochromes, or for structural modifications such as production of F(ab')2 or monvalent Fab fragments. This unit contains protocols for purification of IgG by ammonium sulfate precipitation coupled with size‐exclusion chromatography, Protein A‐ and Protein G‐affinity chromatography, immunoaffinity chromatography, and ion‐exchange chromatography.
Fragmentation of Immunoglobulin G
Authors: Andrew Sarah M. , Titus Julie A.
Published: June 2004

For some purposes, fragments of the IgG molecule are preferred. The Fc portion is useful for studies of biological effect–binding to the Fc receptor, mediating antibody‐dependent cellular cytotoxicity, and complement fixation. The bivalent F(ab')2 produced by digestion with pepsin and the monovalent Fab produced by digestion with papain are useful for studies based on the interaction between antibody binding site(s) with antigen. This unit also describes alternative methods for preparing F(ab')2 fragments.
Antibody Conjugates for Cell Biology
Authors: Haugland Rosaria P.
Published: June 2004

Antibody conjugates are extremely useful reagents for probing many biologically and chemically important molecules in vitro or in vivo. This unit presents some basic protocols for conjugating antibodies with fluorescent dyes, with biotin, and with enzymes. These protocols have been selected because they are relatively easy to perform and have high rates of success. The basic approach for conjugating antibodies is to derivatize amino groups on the antibody with the dye or biotin derivative of choice. Conjugation at amine sites is simple to perform, and, with a fluorescent dye, it generally yields brightly fluorescent conjugates. The conjugates retain high levels of biological activity, notwithstanding the fact that the labels react with random amines on the antibody molecule, and they may even react near or at the antigen‐binding site. Labeling of the amines is the most widely used commercial method to tag antibodies. In this unit, suggestions are presented for variations on these basic protocols that are useful when quantities of available antibody are limited. Methods for estimating antibody concentration are also included.
Production of Antibodies That Recognize Specific Tyrosine‐Phosphorylated Peptides
Authors: DiGiovanna Michael P. , Roussel Robert R. , Stern David F.
Published: June 2004

Phosphorylation of tyrosine residues of various proteins plays an important role in regulation of the functional activities of proteins within the cell. Antibodies to specific tyrosine‐phosphorylated peptides are a valuable tool for analysis of the localization and function of individual phosphoproteins. This unit describes the methods used to produce and screen monoclonal and polyclonal antibodies for specificity for only the phosphorylated state of a peptide.
Imaging Protein‐Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy
Authors: Wouters Fred S. , Bastiaens Philippe I.H.
Published: June 2004

FRET microscopy enables the detection of different biochemical states of proteins in cells. The use of fluorescence in the detection of proteins, by chemical modification, by immunofluorescence, or by genetic encoding of a green fluorescent protein fusion protein, provides more information than just the location of the protein in the cell. The properties of the fluorophore can be exploited to extract information on protein‐protein interactions. A straightforward, quantitative imaging approach is presented to measure FRET that is based on internal calibration by acceptor photobleaching.
Identification of Protein Interactions by Far Western Analysis
Authors: Edmondson Diane G. , Roth Sharon Y.
Published: June 2004

Far Western blotting is a method for detecting protein‐protein interactions. This technique utilizes a nonantibody protein probe to detect interacting proteins immobilized on a membrane support. Proteins to be assayed can be prepared by multiple techniques and detected by several labeling schemes.