bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fluorescence Detection of Glycoproteins in Gels and on Electroblots
Authors: Patton Wayne F. 
Published: June 2004

AbstractGlycosylation of proteins is a dynamic post‐translational modification commonly found in eukaryotes, changing with development, differentiation, and disease state. Identification of glycoproteins is important for completely characterizing the population of proteins found at a given point in time. This unit contains protocols for identifying glycoproteins in general using a fluorescence based periodate/Schiff base method and those containing specific carbohydrates using a lectin‐based fluorescent method.
Metabolic Labeling with Amino Acids
Authors: Bonifacino Juan S. 
Published: June 2004

AbstractMetabolic labeling techniques are used to study the biosynthesis, processing, intracellular transport, secretion, degradation, and physical‐chemical properties of proteins. This unit provides protocols for metabolic labeling of cells with [35S]‐labeled amino acids to provide material suitable for analysis by immunoprecipitation, for characterization of cellular proteins, for analysis of protein trafficking, and for one‐ and two‐dimensional gel electrophoresis. Three procedures are described for suspension and adherent cells: pulse‐labeling, pulse‐chase labeling, or continuous long‐term labeling. A support protocol describes TCA precipitation to measure the extent of labeling.
Metabolic Labeling with Sulfate
Authors: Tooze Sharon A. 
Published: June 2004

AbstractPost‐translational modifications of proteins make it possible to determine where a protein normally resides or to follow its transport through the cell. One such modification is addition of sulfate either to tyrosine residues or to carbohydrate side chains. Labeling studies with [35S] sulfate can be done as continuous or pulse‐chase experiments, as described here.
Metabolic Labeling with Fatty Acids
Authors: Jackson Caroline S. , Magee Anthony I. 
Published: June 2004

AbstractCovalent attachment of radiolabeled fatty acids (e.g., [3H]myristate or palmitate) is an alternative method for labeling proteins. This unit contains methods for biosynthetic labeling with fatty acids, analysis of the fatty acid linkage with protein, analysis of total protein‐bound fatty acid level in cell extracts, and analysis of the identity of the bound fatty acid.
Cell‐Substrate Adhesion Assays
Authors: Humphries Martin J. 
Published: June 2004

AbstractIn the intact organism, cells adhere to a variety of substrates. It is
possible to investigate the nature of the molecules to which cells adhere in
two different assays. In the first protocol presented in this unit, physical
adhesion of a cell is assessed by determining the extent to which the cell
spreads on a defined substrate‐‐the plate is coated with the
test substance, cells are added and allowed time to attach and spread, and
the extent of spreading is assessed using phase contrast microscopy. In a
second assay, an aliquot of cells is added to the well of a microtiter plate
coated with a test adhesion molecule and the cells are allowed to attach.
Nonattached cells are removed, and the percent of added cells attached to the
substrate is quantified by crystal violet staining.
Quantitative Measurement of Cell Adhesion Using Centrifugal Force
Authors: McClay David R. , Hertzler Philip L. 
Published: June 2004

AbstractIn order to study the biophysical forces involved in
cell‐substrate (or cell‐cell) adhesion, it is necessary to
measure the strength of adhesion. Two questions can be addressed using the
centrifugal cell adhesion assay provided in this unit: what is the
ligand‐receptor affinity for cells adhering at 4oC and what
is the strength of the ATP‐dependent processes that strengthen
adhesion at 37oC. In both cases, the strength of adhesion is
measured as the resistance to disruption in the presence of a measured
centrifugal force.
Analyzing Integrin‐Dependent Adhesion
Authors: Mould A. Paul 
Published: June 2004

AbstractThis unit describes methods for the analysis of integrin‐ligand
binding in both cell‐based assays and solid‐phase assays. A
major application of cell adhesion assays is in investigating whether a
certain cell type can adhere to a specific adhesive substrate, and, if so,
which receptors are involved. Particularly if the substrate is a matrix
component (e.g., fibronectin), members of the integrin family are likely to
play a dominant role in adhesion. Procedures are described here for assessing
which integrins are involved in this process. A detailed analysis of ligand
recognition by individual integrins can be performed using a
solid‐phase receptor‐ligand binding assay. The unit also
contains support protocols for integrin purification and coupling of
antibodies to Sepharose for use in this purification, as well as for
biotinylation of integrin ligands to be used in the solid‐phase
assay.
Measurement of Adhesion Under Flow Conditions
Authors: Kucik Dennis F. 
Published: June 2004

AbstractThis unit describes the analysis of dynamic cell adhesion using a flow chamber assay. The flow chamber enables the researcher to reconstruct cell systems in the presence of shear stress to assay adhesion under well&defined forces. These assays are most commonly used to study leukocyte adhesion, either to cultured endothelial cell monolayers or to purified substrates, simulating physiological interactions of leukocytes with endothelial cells. This assay can be also be used to characterize transient adhesive events or adhesion strengthening even for cells that do not normally experience shear stress, because contact time between cells and substrates and anti&adhesive forces can be closely regulated by stopping and starting the flow. Flow chamber assays are also useful for measuring bacterial adhesion under flow.
Purification of Vitronectin
Authors: Akiyama Steven K. 
Published: June 2004

AbstractThis unit describes the purification of extracellular vitronectin from plasma by a series of affinity chromatography steps. First plasma is depleted of fibronectin and other heparin‐binding proteins; the sample is then treated with urea to activate the heparin‐binding activity of vitronectin. The resulting vitronectin is ∼98% pure.
Proteoglycan Isolation and Analysis
Authors: Woods Anne , Couchman John R. 
Published: June 2004

AbstractProteoglycans can be difficult molecules to isolate and analyze due to large mass, charge, and tendency to aggregate or form macromolecular complexes. This unit describes detailed methods for purification of matrix, cell surface, and cytoskeleton‐linked proteoglycans. Methods for analysis of glycoaminoglycan size and type and of core protein species are described.
Matrix Metalloproteinases
Authors: Birkedal‐Hansen Henning , Yamada Susan , Windsor Jack , Poulsen Anne Havemose , Lyons Guy , Stetler‐Stevenson William , Birkedal‐Hansen Bente 
Published: June 2004

AbstractMatrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods‐cell‐mediated dissolution of type I collagen fibrils, direct and reverse zymography, enzyme capture based on a‐2 macroglubulin and TIMP‐1 and ‐2, and demonstration of crytic thiol groups in metalloproteinase precursors‐that are used to characterize the functions of matrix metalloproteinases and their inhibitors.
Purification and Analysis of Thrombospondin‐1
Authors: Yee Karen O. , Duquette Mark , Ludlow Anna , Lawler Jack 
Published: June 2004

AbstractThromboapondin 1 (TSP‐1) is a trimeric matricellular protein that is expressed by many cells. It contains several different domains that allow it to participate in cell adhesion, cell migration, and cell signaling. Recently TSP‐1 has been shown to activate transforming growth factor β (TGF‐β) and to inhibit both angiogenesis and tumor growth. This unit contains protocols for the purification of TSP‐1 from platelet‐rich plasma and the purification of TSP‐1 proteolytic fragments.
In Vitro Analysis of Endoplasmic‐Reticulum‐to‐Golgi Transport in Mammalian Cells
Authors: Allan Bernard B. , Balch William E. 
Published: June 2004

AbstractA temperature‐sensitive mutant of vesicular stomatitis G protein is used to follow the movement of that protein from the endoplasmic reticulum to transport vesicles to cis‐Golgi and finally medial/trans‐Golgi by assessing the maturation of two asparagine‐linked oligosaccharides. These assays can be used to identify the factors that are required for and regulate protein trafficking through these compartments.
In Vitro Analysis of SV40 DNA Replication
Authors: Yin Yi‐Ling , Dutta Anindya 
Published: June 2004

AbstractThis unit describes a method for studying cellular factors that affect eukaryotic replication by using a plasmid that contains an SV40 origin of replication and supplying the viral helicase T antigen. Replication factors are supplied by S100 extracts of 293 cells. The unit also contains protocols for expression and purification of T antigen and analysis of the products of replication.
In Vitro Transcription
Authors: Kamakaka Rohinton T. , Kraus W. Lee 
Published: June 2004

AbstractIn vitro analysis of transcription and the factors that play a role in transcription require preparation of an extract that faithfully reproduces in vivo transcription. This unit describes protocols for generating extracts of mammalian cells and Drosophila embryos that are competent for in vitro transcription reactions using naked DNA or chromatin templates and for primer extension analysis of transcripts.
Analysis of RNA Export Using Xenopus Oocytes
Authors: Duricka Deborah , Ullman Katharine S. 
Published: June 2004

AbstractThis unit describes a procedure for monitoring RNA export in Xenopus oocytes. The technique involves synthesizing labeled RNA in vitro and microinjecting the RNA into oocyte nuclei. Following incubation the oocytes are dissected into nuclear and cytoplasmic fractions. These samples are then processed for RNA analysis, allowing the extent of export to be quantitatively assessed.
Cell Wound Assays
Authors: McNeil Paul L. , Clarke Mark F.S. , Miyake Katsuya 
Published: June 2004

AbstractMany mammalian and invertebrate cells that reside in mechanically active tissue environments normally and frequently experience disruptions in plasma membrane integrity which are rapidly repaired and do not lead to cellular death. This unit describes methods for identifying “wounded” cells, often present as a minority in culture or tissues. The use of these methods can provide information about the frequency and extent of cellular wounding.
Optical Microscopy–Based Migration Assay for Human Neutrophils
Authors: Pierini Lynda M. , Maxfield Frederick R. 
Published: June 2004

AbstractThis unit describes an in vitro microscopy assay for examining the migration of human neutrophils in two dimensions to identify the underlying cause of a migration defect and to evaluate a variety of migration parameters that cannot be studied using migration through a porous filter. Freshly isolated human neutrophils a placed in the chamber, stimulated, and images are collected at various time points. The data can be used to determine the effects of a pharmacological treatment on the migration of individual cells.
In Vitro Motility Assay to Study Translocation of Actin by Myosin
Authors: Sellers James R. 
Published: June 2004

AbstractA basic property of myosin is its ability to interact with and translocate actin. This unit describes an in vitro motility assay that can be used to study the translocation, or sliding, of actin filaments by myosin bound to a coverslip. The assay makes use of the ability to image single F‐actin filaments labeled with rhodamine phalloidin, a high‐affinity fluorescent ligand using fluorescence microscopy. The system is fast, easy to set up and maintain, uses only small amounts of protein, and yields quantitative results.
Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
Authors: Hawes Chris , Brandizzil Federica , Batoko Henri , Moore Ian 
Published: June 2004

AbstractBy the addition of plant or mammalian targeting sequences, green fluorescent protein (GFP) can be directed to the endoplasmic reticulum, the Golgi apparatus, or both organelles in plant cells. This unit describes the application of rapid Agrobacterium and virus‐mediated transient expression systems in leaf tissue to permit visualization of ER and Golgi dynamics in vivo by epifluorescence or confocal microscopy.
Overview of Protein Trafficking in the Secretory and Endocytic Pathways
Authors: Enns Caroline 
Published: June 2004

AbstractProteins migrate from the site of their synthesis to intracellular, surface, or extracellular sites depending on their function. This well‐illustrated overview provides an introduction to the pathways of protein trafficking. It includes descriptions of the relevant cellular compartments, modifications associated with the movement of proteins through these compartments, signals used for targeting and translocation, mechanisms for transit between compartments, and methods for sorting proteins.
Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
Authors: Francis Edwin , Daniels Robert , Hebert Daniel N. 
Published: June 2004

AbstractProteins that travel through the secretory pathway undergo post‐translational folding and oxidation steps that lead to correct conformation of the final protein. This unit focuses on methods for the analysis of folding and oxidation events and the factors responsible for their proper execution. Alkylation and nonreducing SDS‐PAGE is use to analyze disulfide bond formation in ER‐derived microsomes. If proteins are synthesized under reducing conditions, it is possible to initiate folding by addition of oxidizing agents, thus allowing analysis of factors necessary for the folding process. As folding progresses, the protein of interest shows a change in sensitivity to proteolysis. Co‐immunoprecipitation or crosslinking and denaturing immunoprecipitation are used to explore the role of molecular chaperones and other factors. Conformation‐specific antibodies can be used to probe folding. In addition, folding can be analyzed in intact or semi‐permeabilized adherent or suspension cells.
Analysis of Caspase Activation During Apoptosis
Authors: Kaufmann Scott H. , Kottke Timothy J. , Martins L. Miguel , Henzing Alexander J. , Earnshaw William C. 
Published: June 2004

AbstractThis unit describes three methods for the detection of caspase activation as cells undergo apoptosis. Simple and relatively quantitative enzymatic assays are provided using suitable substrates. Because the various low‐molecular‐weight substrates available for these assays are not selective, however, the assays do not accurately distinguish between various caspases. Immunoblotting is described for following the activation of specific caspases. When coupled with subcellular fractionation, this method can provide large amounts of temporal and spatial information about caspase activation. Finally, affinity labeling protocols are provided for detecting active caspases in whole‐cell lysates or subcellular fractions.
Analysis of Mitochondrial Dysfunction During Cell Death
Authors: Gogvadze Vladimir , Orrenius Sten , Zhivotovsky Boris 
Published: June 2004

AbstractAttempts to identify a common underlying step in the apoptotic program in response to various cytotoxic stimuli have focused on the role of mitochondria in this form of cell death. This unit contains a family of protocols that can be used to assess mitochondrial functions during apoptotic responses. Protocols are included for the collection and analysis of released proteins, for detection of the mitochondrial permeability transition, for measurement of mitochondrial membrane potential, and for preparation of mitochondria from different tissue sources.
Tail Vein Assay of Cancer Metastasis
Authors: Elkin Michael , Vlodavsky Israel 
Published: June 2004

AbstractThe most damaging change during cancer progression is the switch from a locally growing tumor to a metastatic killer. This switch involves numerous alterations that allow tumor cells to complete the complex series of events needed for metastasis. In considering steps required for successful metastasis, extravasation from blood vessels in target organs is regarded as a critical process. Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining of blood vessels and degrade its underlying basement membrane in order to escape into the extravascular tissue where they establish metastasis. This unit describes the most common assay applied to evaluate the metastatic potential of blood‐borne tumor cells. The protocol is often called “experimental metastasis”, distinct from “spontaneous metastasis”, where the tumor cells are first allowed to form a primary tumor in the site of injection and then escape into lymphatic or blood circulation. Cultured tumor cells are injected into the tail vein and allowed to circulate. After 12 to 20 days the recipients are euthanized and the lungs are evaluated for the presence of metastatic tumors.
SAGE Analysis from 1 µg of Total RNA
Authors: Cai Jerry , Ash David , Jabs Ethylin Wang 
Published: June 2004

AbstractSerial analysis of gene expression (SAGE) is a powerful transcription‐profiling method that allows simultaneous expression analysis of thousands of transcripts, provides absolute digital readout of the expression level, and identifies new genes. A disadvantage of SAGE is the relatively high amount of input RNA required. Consequently, several techniques have been developed to overcome this limitation, so that SAGE can be applied to very limited amounts of starting material, e.g., small biological samples such as tissue biopsies or microdissected materials. Here we describe a modified version of the original microSAGE protocol, which requires only 1 to 2 ug total RNA. This method avoids PCR amplification of cDNA and reamplification of ditags, procedures that potentially compromise the quantitative nature of this technique.
The Importance of Biological Databases in Biological Discovery
Authors: Baxevanis Andreas D. 
Published: June 2004

AbstractBiological databases play a central role in bioinformatics. They offer scientists the opportunity to access sequence and structure data for tens of thousands of sequences from a broad range of organisms. This unit provides a brief overview of some existing sequence databases, such as GenBank, UCSC Genome Browser, and Ensembl. It also discusses non‐sequence centric databases, such as OMIM.
Using the NCBI Map Viewer to Browse Genomic Sequence Data
Authors: Wolfsberg Tyra G. 
Published: June 2004

AbstractThis unit includes an introduction to the Map Viewer, which describes how to perform a simple text‐based search of the genome to view the genomic context of a gene, navigate along the chromosome, zoom in and out, and change the displayed maps to hide and show information. It also describes some of NCBI's sequence‐analysis tools, which are provided as links from the Map Viewer. The Alternate Protocols describe different ways to query the genome sequence, and also illustrate additional features of the Map Viewer. shows how to perform and interpret the results of a BLAST search against the human genome. demonstrates how to retrieve a list of all genes between two STS markers. Finally, shows how to find all annotated members of a gene family.
Using the Blocks Database to Recognize Functional Domains
Authors: Henikoff Jorja G. , Greene Elizabeth A. , Taylor Nick , Henikoff Steven , Pietrokovski Shmuel 
Published: June 2004

AbstractBlocks are ungapped multiple alignments of related protein sequence segments that correspond to the most conserved regions of the proteins. The Blocks Database is a collection of blocks representing known protein families that can be used to compare a protein or DNA sequence with documented families of proteins. Protocols in this unit describe the analysis of proteins and families using Blocks‐based tools, including searching, exploring relationships with trees, making new blocks, and designing PCR primers from blocks for isolating homologous sequences.
Identifying Protein Domains with the Pfam Database
Authors: Finn Robert , Griffiths‐Jones Sam , Bateman Alex 
Published: June 2004

AbstractPfam is a database of such protein domain families, with each family represented by multiple sequence alignments and profile hidden Markov models (HMMs). In addition, each family has associated annotation, literature references and links to other databases. The entries in Pfam are available via the worldwide web and in flatfile format. This unit contains detailed information on how to access and utilise the information present in the Pfam database, namely the families, multiple alignments and annotation. Details on running Pfam, both remotely and locally are presented.
Finding Homologs to Nucleic Acid or Protein Sequences Using the Framesearch Program
Authors: Healy Matthew 
Published: June 2004

AbstractThe Framesearch algorithm includes the possibility of a frameshift error in its alignment algorithm, and therefore can find alignments that span different reading frames. Protocols in this unit describe the use of Framesearch to search a protein sequence database for sequences that are similar to a query nucleotide sequence, and to search a nucleotide sequence database for sequences that are similar to a query protein sequence. Three alternate protocols describe ways to improve the speed of Framesearch and thus make it practical for routine use. Framesearch is especially appropriate for low‐quality single‐read nucleotide sequence data, such as ESTs (expressed sequence tags) or early drafts of genomic sequences; it does not offer any significant advantage over less CPU‐intensive algorithms for relatively high‐quality nucleotide sequences without many single‐nucleotide insertion or deletion errors.
Finding Homologs in Amino Acid Sequences Using Network BLAST Searches
Authors: Ladunga Istvan 
Published: June 2004

AbstractBLAST, Basic Local Alignment Search Tool is used more frequently than any other biosequence database search program. The purpose of this unit is not only to show how to run searches on the Web, but also to demonstrate how to fine‐tune arguments for a specific research project. It also offers guidance for interpreting results, handling statistical significance and biological relevance issues, and selecting complementary analyses. This unit covers three classes of the BLAST program: standard protein‐to‐protein searches, translated searches when either the query or the database consists of nucleotide sequences translated into proteins, and finally programs for comparing two sequences (as opposed to searching one sequence against a database of sequences).
Selecting the Right Protein‐Scoring Matrix
Authors: Wheeler David 
Published: June 2004

AbstractEvery program for searching protein sequences against a database includes a choice of a protein weight matrix, also called a scoring matrix. Weight matrices add sensitivity to the search, while statistical significance adds selectivity. Virtually every user chooses the default, typically PAM 250 or BLOSUM62. Despite the fact that the choice of matrix can strongly influence the outcome of the analysis, most users do not know why a particular matrix should be used. In general, scoring matrices implicitly represent a particular theory of protein sequence evolution. Understanding the assumptions underlying the PAM and BLOSUM scoring matrices can aid in making the proper choice. The purpose of this unit is to guide the choice of a scoring matrix. It covers the selection of PAM matrices, BLOSUM matrices and provides a brief overview of the wide variety of specialized scoring matrices.