bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assays for CDK Activity and DNA Replication in the Cell Cycle
Authors: Pines Jonathon , Jackman Mark , Simpson Karen 
Published: June 2004

AbstractThis 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

AbstractExponentially 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

AbstractThis 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

AbstractDifferential 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

AbstractThis 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

AbstractThe 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

AbstractThis 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

AbstractGelled 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

AbstractThis 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

AbstractFibroblasts 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

AbstractSPARC 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

AbstractThe 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

AbstractThe 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

AbstractRough 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

AbstractImport 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

AbstractA 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

AbstractMuch 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

AbstractIn 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

AbstractIn 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

AbstractIn 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.
In Vitro Analysis of Peroxisomal Protein Import
Authors: Terlecky Stanley R. 
Published: June 2004

AbstractThis 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

AbstractChemotaxis 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

AbstractBasement 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.
Microtubule/Organelle Motility Assays
Authors: Waterman‐Storer Clare M. 
Published: June 2004

AbstractThis 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.
Measuring Dynamics of Nuclear Proteins by Photobleaching
Authors: Dundr Miroslav , Misteli Tom 
Published: June 2004

AbstractThe 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

AbstractPhosphorylation 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

AbstractIncorporation 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

AbstractElucidation 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

AbstractProteins 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

AbstractThis 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

AbstractMany 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

AbstractSpatial 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.