bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amplification Using CHO Cell Expression Vectors
Authors: Kingston Robert E. , Kaufman Randal J. , Bebbington C.R. , Rolfe M.R. 
Published: June 2004

AbstractThe ability to select for integration of plasmid DNA into the host chromosome allows the generation of stably transfected cell lines. With transfection of a selectable marker linked to a nonselectable target gene (or by cotransfection of the two unlinked genes), high‐level expression of the desired gene is obtained by selecting for amplification of the selectable marker. This unit presents two systems for gene amplification and expression. The first describes the dihydrofolate reductase (DHFR) selection system while the second is based on selection of the glutamine synthetase (GS) gene. The DHFR system is probably more widely used, and results in very high levels of amplification and expression; however, the DHFR amplification process is lengthy and may require several months to isolate and characterize a stable, amplified line. In contrast, the GS system typically requires only a single round of selection for amplification to achieve maximal expression levels.
Chemical Labeling of Carbohydrates by Oxidation and Sodium Borohydride Reduction
Authors: Fukuda Minoru 
Published: June 2004

AbstractThis unit describes a collection of methods for chemical labeling of carbohydrates (free oligosaccharides or oligosaccharides conjugated to proteins, peptides, or lipids) by oxidation followed by reduction or by direct reduction. Oligosaccharides can be labeled with either radioisotopes or nonradioactive fluorescent molecules. These labelings allow one to follow the oligosaccharides during chromatography and in cells if labeled by fluorescent molecules. Selective oxidation with mild periodate followed by reduction with tritiated sodium borohydride results in selective radiolabeling of sialic acid residues on oligosaccharides or glycoproteins. Alternatively, treatment of samples with galactose oxidase results in oxidation of galactose or N‐acetylgalactosamine residues at nonreducing termini, rendering these residues suceptible to labeling with NaB[3H]4. Oxidized glycoconjugates can also be labeled using the fluorescent probe lucifer yellow CH. Free oligosaccharides can be labeled by reduction with NaB[3H]4. An additional protocol describes the release and simultaneous labeling of O‐glycan oligosaccharides by alkaline beta‐elimination in the presence of NaB[3H]4.
Sialidases
Authors: Powell Leland D. , Varki Ajit P. 
Published: June 2004

AbstractSialic acids are a family of nine‐carbon acidic sugars found at the nonreducing terminus of many glycoconjugates. Sialidases can remove these sugar units selectively from cell surfaces, membranes, or purified glycoconjugates. In this unit, sialidase digestion of purified glycoproteins is described as is treatment of intact cells. The physical properties of the four most useful sialidases are discussed along with their relative activities against sialic acids with different modifications and in different linkages
Endoglycosidase and Glycoamidase Release of N‐Linked Oligosaccharides
Authors: Freeze Hudson H. 
Published: June 2004

AbstractCarbohydrate chain modifications are often used to monitor glycoprotein movement through the secretory pathway. This is because stepwise sugar‐chain processing is unidirectional and generally corresponds to the forward or anterograde movement of proteins. This unit offers a group of techniques that will help analyze the general structure of carbohydrate chains on a protein and, therefore, oligosaccharide processing mileposts. The sugar chains themselves are not analyzed, but their presence and structure are inferred from gel mobility differences after one or more enzymatic digestions. This approach is most often used in combination with
[35S]Met pulse‐chase metabolic labeling protocols, but they can be applied to any suitably labeled protein (e.g., biotinylated or
125I‐labeled).
Preparation of Glycopeptides
Authors: Powell Leland D. 
Published: June 2004

AbstractGeneration of glycopeptides from glycoproteins is frequently useful when analyzing a protein's oligosaccharide side chains. Freed from the bulk of the polypeptide backbone by proteolysis, glycopeptides can be characterized by a variety of techniques. Extensive proteolysis with pronase or proteinase K results in oligosaccharides with one or a few amino acid residues attached. This technique, detailed in this unit, is often employed as a first step in characterizing oligosaccharides on very large glycoproteins such as proteoglycans and mucins. Limited proteolysis with a specific endoproteinase (e.g., trypsin, a‐chymotrypsin, and V8 protease) is also described, and leaves a larger peptide attached to the oligosaccharide. The resulting glycopeptides are generally suitable substrates for Peptide:N‐glycosidase F, an enzyme useful in defining oligosaccharide‐peptide linkages. Additionally, they can be separated by reversed‐phase chromatography, resulting in a glycopeptide map that is analogous to a peptide map, and used for detection of glycosylation sites.
Composition of Labeled Monosaccharides from Glycosaminoglycans
Authors: Conrad H. Edward 
Published: June 2004

AbstractProteoglycans can be metabolically labeled with [3H]glucosamine to incorporate label into glucosamine and galactosamine. Glycosaminoglycans can be released from the proteoglycans and the distribution of labeled glucosamine and galactosamine in the isolated glycosaminoglycans can be determined. A series of acid treatments yields a mixture of disaccharides (uronosyl‐anhydro sugars), free uronic acids, and free anhydro sugars, which are then reduced and separated by paper chromatography. The amount of each labeled component is quantified by scintillation counting. This procedure can also be used to determine the composition of glycosaminoglycans that have not been labeled metabolically. Samples of unlabeled glycosaminoglycans can be treated in the same manner and the hydrolysis/deamination mixture can be reduced with NaB3H4 to give stoichiometric 3H labeling of each reducing sugar component of the mixture.
Analysis of Phosphorylation of Unlabeled Proteins
Authors: Sefton Bartholomew M. 
Published: June 2004

AbstractPhosphorylation of unlabeled proteins can be detected using immunological or enzymatic techniques. Anti‐phosphotyrosine antibodies are used with immunoblots to detect tyrosine phosphorylation. This unit provides a protocol for detection of antibodies by 125I‐labeled protein A or enhanced chemiluminescence (ECL). Detection of enzymatic dephosphorylation using a general or phosphoamino acid‐specific phosphatase and subsequent SDS‐PAGE mobility shift (or a change in activity) is also described.
Production of Antibodies That Recognize Specific Tyrosine‐Phosphorylated Peptides
Authors: DiGiovanna Michael P. , Roussel Robert R. , Stern David F. 
Published: June 2004

AbstractIt is possible to produce anti‐phosphopeptide antibodies (i.e., antibodies recognizing phosphorylated peptides) that recognize a protein only in its phosphorylated state, and that do not cross‐react with either the cognate unphosphorylated protein or other phosphoproteins. Unlike conventional antibodies, anti‐phosphopeptide antibodies provide information regarding not only the abundance of a protein but also its activity. Also, unlike general anti‐phosphoamino acid (e.g., anti‐phosphotyrosine) antibodies, which have broad reactivity, anti‐phosphopeptide antibodies may have unique specificity toward the cognate proteins. Such reagents not only facilitate conventional in vitro analysis of phosphoproteins, but also allow heretofore impossible applications, e.g., differential isolation of species of a particular protein that have been phosphorylated at individual phosphorylation sites, as well as analysis of the functional state of a protein in situ by immunohistochemical techniques. This unit provides protocols for the production of both polyclonal and monocloncal anti‐phosphopeptide antibodies. Support protocols are provided for the coupling of peptides and phosphotyrosine to the affinity matrix (Affi‐Gel 10); BSA‐agarose affinity matrix is commercially available.
Sequence Similarity Searching Using theBLASTFamily of Programs
Authors: Wolfsberg Tyra G. , Madden Thomas L. 
Published: June 2004

AbstractDatabase sequence similarity searching is carried out thousands of times each day by researchers worldwide and has become a very valuable tool. Over the years, a number of algorithms have been implemented to facilitate database searching. The BLAST (Basic Local Alignment Research Tool) family of sequence similarity search programs allows searches to be done quickly and easily, but with sensitive, yet rigorous statistical expectations. In this unit, which is a completely new version of its predecessor of the same title, the user learns how to access the databases, determine the correct searching strategies, and apply examples of BLAST searches to his or her own data.
Separation of Histone Variants and Post‐Translationally Modified Isoforms by Triton/Acetic Acid/Urea Polyacrylamide Gel Electrophoresis
Authors: Ryan Colleen A. , Annunziato Anthony T. 
Published: June 2004

AbstractDue to their similarities in size and charge, complete resolution of histones by electrophoresis poses a considerable challenge. The addition of nonionic detergents to the traditional acetic acid/urea (AU) polyacrylamide gel electrophoresis (PAGE) system has afforded an excellent method to separate not only the different modified forms of histones, but also the primary sequence variant subtypes of selected histone species; it is widely used to separate histones with varying levels of acetylation. This unit describes the use of gels containing the nonionic detergent Triton X‐100, referred to as Triton/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones. Also included are support protocols detailing several accessory techniques: assembly of gel plates for the TAU gel, preparation of histones from isolated nuclei in a solubilized form amenable to electrophoresis, and electrophoretic transfer of proteins from these gels to PVDF membranes.
Chromatin Assembly Using Drosophila Systems
Authors: Fyodorov Dmitry V. , Levenstein Mark E. 
Published: June 2004

AbstractTo successfully study chromatin structure and activity in vitro, it is essential to have a chromatin assembly system that will prepare extended nucleosome arrays with highly defined protein content that resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. The Drosophila ATP‐dependent chromatin assembly system described in this unit meets these requirements. The end product of the reaction described here has highly periodic extended arrays with physiologic spacing and positioning of the nucleosomes.
Production of a Subtracted cDNA Library
Authors: Klickstein Lloyd B. 
Published: June 2004

AbstractFor some experiments, a complete cDNA library is unnecessary and instead, a subtracted cDNA library is useful. A subtracted cDNA library contains cDNA clones corresponding to mRNAs present in one cell or tissue type and not present in a second type. This cDNA library is used to isolate a set of cDNA clones corresponding to a class of mRNAs, or to aid in the isolation of a cDNA clone corresponding to a particular mRNA where the screening procedure for the cDNA clone is laborious because a specific DNA or antibody probe is unavailable. Since relatively few recombinants are obtained after subtraction, this protocol is for a cDNA library constructed in the λ>10 vector or its equivalent, which allows a high cloning efficiency and permits elimination of nonrecombinants; however, the protocol can be used to produce subtracted cDNA libraries in any vector system.
AFLP‐Based Transcript Profiling
Authors: Vos Pieter , Stanssens Patrick 
Published: June 2004

AbstractThis unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP‐fingerprinting of double‐stranded cDNA. The protocol described includes the following steps: the isolation of poly(A)+ RNA from total RNA, the synthesis of double‐stranded cDNA, the preparation of template fragments by digestion of the cDNA library with a combination of two restriction enzymes and the ligation of adaptors to the fragment ends, the selective amplification of specific subsets of fragments, and the electrophoretic analysis of these amplification products on standard denaturing polyacrylamide gels. The transcript profiles obtained by this technique are a reliable and efficient tool to identify differentially expressed mRNAs.This unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP‐finger his unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP‐finger
Gene Expression Analysis of a Single or Few Cells
Authors: Klein Christoph A. , Zohlnhöfer Dietlind , Petat‐Dutter Karina , Wendler Nicole 
Published: June 2004

AbstractThe need to analyze rare cells is based on the nature of tissue differentiation and regeneration, the initiation and propagation of disease processes in multicellular organisms, and the functional diversity of individual cells. Gene transcription is the most important regulatory mechanism by which a phenotype and functional state of a cell is determined. Therefore, procedures for the qualitative and quantitative assessment of mRNA abundance are important. This unit presents a protocol for semi‐quantitative analysis of gene expression of a single cell and quantitative representation of expressed genes from >10 to 30 cells. A basic protocol for array hybridization on nylon filters is provided because such filters are available in every laboratory. Tissue samples contain many different cell types in variable amounts, so their analysis may require microdissection; a protocol for obtaining cryosections is given. Finally, a simple procedure to prepare the data for statistical analysis is also provided.
Agarose Gel Separation/Isolation of RNA‐Protein Complexes
Authors: Lee Chung‐Sheng Brian , Das Rita , Reed Robin 
Published: June 2004

AbstractIn many methods currently used to analyze RNA‐protein complexes, high salt or other stringent treatments are required for reducing nonspecific interactions and resolving the complex of interest. RNA‐protein complexes often dissociate on native polyacrylamide gels and can only be detected on density gradients or by gel filtration. Agarose gel electrophoresis provides an alternative method that is simple, rapid, and can have high resolution of RNA‐protein complexes. Moreover, the use of low‐melting point agarose for the fractionation readily allows for the isolation of the RNA species in each complex detected on the native gel.
Commonly Used Reagents and Equipment
Authors: Moore David D. 
Published: June 2004

AbstractThis appendix lists recipes for common reagents and also describes equipment that is typically found in a molecular biology laboratory.
Buffers and Stock Solutions
Acid precipitation solution
Ammonium acetate, 10 M
BBS (BES‐buffered solution), 2×
CaCl2, 1 M
Denhardt solution, 100×
Dithiothreitol (DTT), 1 M
EDTA (ethylenediamine tetraacetic acid), 0.5 M (pH 8.0)
Ethidium bromide, 10 mg/ml
HBSS (Hanks balanced salt solution)
HCl, 1 M
HeBS (HEPES‐buffered saline) solution, 2×
KCl, 1 M
MgCl2, 1 M
MgSO4, 1 M
MOPS buffer
NaCl, 5 M
NaOH, 10 M
PBS (phosphate‐buffered saline)
Potassium acetate buffer, 0.1 M
Potassium phosphate buffer, 0.1 M
SDS electrophoresis buffer, 5×
SED (standard enzyme diluent)
Sodium acetate, 3 M
Sodium acetate buffer, 0.1 M
Sodium phosphate buffer, 0.1 M
SSC (sodium chloride/sodium citrate), 20×
STE buffer
TAE (Tris/acetate/EDTA) electrophoresis buffer
TBE (Tris/borate/EDTA) electrophoresis buffer
TE (Tris/EDTA) buffer
TEA (triethanolamine) solution
TEN (Tris/EDTA/NaCl) solution
TM buffer, 10×
Tris‐buffered saline (TBS)
Tris⋅Cl [tris(hydroxymethyl)aminomethane], 1 M
TTBS (Tween 20/TBS)
Dialysis and Ultrafiltration
Authors: Zumstein Louis 
Published: June 2004

AbstractThe first section of this unit describes dialysis of large‐ and small‐volume samples using cellulose membranes with pore sizes designed to exclude molecules above a selected molecular weight. A support protocol describes preparation of membranes for dialysis. The second section describes protocols for ultrafiltration, and the third section describes simple methods of concentrating solutions.
Statistics for the Molecular Biologist: Group Comparisons
Authors: Ryder Elizabeth F. , Robakiewicz Phil 
Published: June 2004

AbstractIn this appendix, some of the statistical tests most commonly used (and misused) in biological research are discussed. These tests are used for comparisons among groups (e.g., t test and ANOVA). A number of other important areas (e.g., linear regression, correlation, and goodness‐of‐fit testing) are not covered. The purpose is to enable the reader to determine rapidly the most appropriate way to analyze data, and to point out some of the most common errors to avoid.
Induction and Measurement of Cytotoxic T Lymphocyte Activity
Authors: Wonderlich John , Shearer Gene , Livingstone Alexandra 
Published: June 2004

AbstractCytotoxic T lymphocytes (CTL) kill target cells on the basis of cell‐surface antigen recognition and are important in the host response to tumors, transplants, and viruses. This unit presents several protocols for generating and measuring CTL activity. The first describes generating CTL against some of the most commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. In the chromium‐release assay provided here, labeled antigenic targets are recognized and lysed, releasing radioactivity into the supernatant. In the JAM test protocol, CTL activity is determined by measuring degradation of radioactively labeled DNA in target cells that have undergone apoptotic cell death. Rather than measuring release of radioactivity, the JAM test measures the amount of DNA retained in target cells that are not killed by CTL. Two support protocols detail the generation of CTL precursors (CTLp) against antigens that require priming in vivo. A second set of support protocols describe the preparation of both stimulator and target cells for these responses using two representative antigens, trinitrophenyl and viruses. Finally, two alternate protocols illustrate how to determine total CTLp activity in a population that might express cytolytic activity. These protocols bypass MHC restriction and the original antigen specificity of CTLp by polyclonal stimulation of CTLp with mitogens followed by attachment of CTL to target cells and subsequent cytolysis.
Blood Collection
Authors: Donovan John , Brown Patricia 
Published: June 2004

AbstractBlood is most frequently sampled for evaluation of serum antibodies or analysis of surface markers on peripheral blood cells. The protocols offered in this unit describe collection of blood from the orbital sinus or plexus of the mouse, rat, or hamster. With appropriate techniques, small amounts of blood can be obtained with little ill effect on the animal. Collection from the auricular vein or artery of the rabbit is also relatively unstressful to the animal. Bleeding procedures that should be performed on the anesthetized animal include collection from the mouse axillary plexus, cardiac puncture of the mouse, rat, hamster, or rabbit, and collection from the hamster abdominal aorta or vena cava.
Euthanasia
Authors: Donovan John , Brown Patricia 
Published: June 2004

AbstractEuthanasia of laboratory animals must be performed by trained personnel using appropriate techniques, equipment, and reagents in order to effect a death that is humane and satisfies research requirements. Acceptable methods of euthanasia are painless or minimize distress, and are quick and easy to perform, safe for those performing the procedure, and efficient and economic. They are aesthetically acceptable and are done in the absence of other animals. In addition, these methods do not result in gross histological or histochemical changes that would adversely affect research results. This unit offers protocols for euthanasia employing carbon dioxide asphyxiation, pentobarbital overdose, exsanguination, and cervical dislocation for the mouse, rat, hamster, and rabbit.
Enzyme‐Linked Immunosorbent Assays
Authors: Hornbeck Peter 
Published: June 2004

AbstractThis unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell‐surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid‐phase reactants. In the first four protocols, solid‐phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid‐phase reactants are cell‐associated molecules. In all protocols, the solid‐phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. The can be used to optimize the different ELISAs. The second support protocol presents a method for preparing alkaline phosphatase conjugates.
Purification of Human IgA
Authors: Pack Todd D. 
Published: June 2004

AbstractThis unit describes the purification of human immunoglobulin A (IgA).The main method utilizes an IgA‐binding protein (IgA‐bp) as an affinity reagent, similar to the IgG‐binding proteins, protein A and protein G. An alternate protocol describes a method for isolating IgA1 using affinity column chromatography with Jacalin, an IgA1‐specific lectin. Another alternate protocol describes IgA purification by conventional fractionation using size‐exclusion chromatography.
Preparation of Immune Stimulating Complexes (ISCOMs) as Adjuvants
Authors: Mowat Allan McI. , Reid George 
Published: June 2004

AbstractPurified proteins are often poorly immunogenic and in such cases the induction of primary immune responses requires use of an adjuvant. The immune stimulating complex (ISCOM) has a unique ability to provoke a full range of immune response to protein antigens, after both parenteral and oral immunization. This unit describes techniques for incorporating proteins into the ISCOM structure, a process that requires the presence of exposed hydrophobic regions on the protein. The basic protocol outlines a method for preparation of ISCOMs containing inherently nonhydrophobic proteins, to which palmitic acid has been attached covalently. Two alternate protocols are given that do not require covalent modification of the protein. In the first, hydrophobic groups are revealed by acid treatment of the protein. The second describes preparation of ISCOMs containing integral membrane proteins that therefore possess a hydrophobic transmembrane domain.