bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conjugation of Enzymes to Antibodies
Authors: Winston Scott E. , Fuller Steven A. , Evelegh Michael J. , Hurrell John G.R. 
Published: June 2004

AbstractConjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme [e.g., horseradish peroxidase (HRPO), urease, or alkaline phosphatase] and an antigen‐specific monoclonal or polyclonal antibody in which neither the antigen‐combining site of the antibody nor the active site of the enzyme is functionally altered. This unit describes procedures for cross‐linking HRPO, urease or alkaline phosphatase to immunoaffinity‐purified monoclonal or polyclonal antibodies (IgG).
Isotype Determination of Antibodies
Authors: Hornbeck Peter , Fleisher Thomas A. , Papadopoulos Nicholas M. 
Published: June 2004

AbstractFrequently it is necessary to know the amount and serological class of antibodies made by an immunized animal, produced by hybridomas, or present in the serum of patients with inflammatory or neoplastic conditions. The immunologist's approach to such a problem is to consider the antibody or immunoglobulin molecules themselves as antigens and to use anti‐immunoglobulin antibodies as the specific and sensitive agents of detection. This unit describes two methods for measurement and classification of supernatant or serum immunoglobulins: an ELISA and a method employing electrophoresis and immunofixation.
Preparation of Myeloma Cells
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R. 
Published: June 2004

AbstractIn this unit, myeloma cells are cultured to ensure their sensitivity to the HAT selection medium used after cell fusion. Cell culture conditions are adjusted such that the Sp2/0 cells are in the log phase of growth and exhibit high viability at the time of collection for fusion. A support protocol is provided to determine the number of viable cells present in the cell culture.
Preparation of Mouse Feeder Cells for Fusion and Cloning
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R. 
Published: June 2004

AbstractTo maximize the yield of hybrids from fusion and cloning procedures, feeder cells are required to be cocultured with the hybrids, while hybrid cell density is low. Mouse peritoneal cells, most of which are macrophages, have been found to be convenient and effective feeder cells which are a source of soluble growth factors for hybridoma cells.
Fusion of Myeloma Cells with Immune Spleen Cells
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R. 
Published: June 2004

AbstractLiquid nitrogen storage is the method of choice for long‐term safekeeping of hybridoma cell lines. Frozen aliquots of originally isolated hybridomas provide insurance against loss of antibody production and vigor during culture. There are many variations of cell freezing methods in use. However, for freezing and recovering hybridomas and lymphoid cells in general, the protocol described in this unit is simple and has been successful.
Determination of the Specific Antibody Titer
Authors: Cooper Helen M. , Paterson Yvonne 
Published: June 2004

AbstractThe amount of specific antibody present in polyclonal antiserum, ascites fluid, or hybridoma supernatant can be quantitated by either solid‐phase radioimmunoassay (RIA) or by direct ELISA. In the solid‐phase assay described here, serially diluted antiserum is incubated in microtiter wells previously coated with the relevant antigen. Bound antibody is detected by employing 125I‐labeled anti‐immunoglobulin antibodies. The amount of specific antibody in the antiserum is then determined from a standard curve generated with a specific antibody of known concentration. The unknown antiserum and the standard antibody are assayed in parallel. The support protocols describe the chloramine T and IODO‐GEN procedures for radioiodination of the anti‐immunoglobulin reagent. The use of the solid‐phase RIA procedure to determine the light‐chain and heavy‐chain isotypes present in polyclonal antisera and fluids containing monoclonal antibodies is also described.
Genome‐Wide Transposon Mutagenesis in Yeast
Authors: Kumar Anuj , Snyder Michael 
Published: June 2004

AbstractThis unit provides comprehensive protocols for the use of insertional libraries generated by shuttle mutagenesis. From the basic protocol, a small aliquot of insertional library DNA may be used to mutagenize yeast, producing strains containing a single transposon insertion within a transcribed and translated region of the genome. This transposon‐mutagenized bank of yeast strains may be screened for any desired mutant phenotype. Alternatively, since the transposon contains a reporter gene lacking its start codon and promoter, transposon‐tagged strains may also be screened for specific patterns of gene expression. Strains of interest may be characterized by vectorette PCR (protocol provided) in order to locate the precise genomic site of transposon insertion within each mutant. A method by which Cre/lox recombination may be used to reduce the transposon in yeast to a small insertion element encoding an epitope tag is described. This tag serves as a tool by which transposon‐mutagenized gene products may be analyzed further (e.g., localized to a discrete subcellular site).
Preparation of Yeast DNA
Authors: Hoffman Charles S. 
Published: June 2004

AbstractMolecular studies in yeast often require the isolation of both plasmid and chromosomal yeast DNA. Plasmid DNA is used in the transformation of E. coli, whereas chromosomal DNA is used for Southern hybridization analysis, in vitro amplification by the polymerase chain reaction (PCR), or cloning of integrated plasmids. This unit presents two variations of the “smash and grab” protocol that produce suitable DNA for all these applications. These protocols work for both Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Preparation of Yeast RNA
Authors: Collart Martine A. , Oliviero Salvatore 
Published: June 2004

AbstractThis unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first protocol isolates RNA directly from intact yeast cells by extraction with hot acidic phenol. This yields RNA that is relatively free of contaminating DNA, is convenient to perform with multiple samples, and gives little or no sample‐to‐sample variation. In contrast, an alternate protocol relies upon disruption of cells by vigorous mixing with glass beads and denaturing agents. Although this procedure results in efficient breaking of the cells, the product is associated with residual DNA, and the procedure itself is troublesome when one is working with multiple samples. A second alternate protocol describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation.
S. pombe Strain Maintenance and Media
Authors: Forsburg Susan L. 
Published: June 2004

AbstractFission yeast can be grown and maintained using similar culture methods to those employed for budding yeast. The optimal media for S. pombe is somewhat different than that employed for S. cerevisiae, although budding yeast media can be used if necessary. Good sterile technique is essential to prevent contamination by airborne molds or bacteria.
In situ Hybridization and Detection Using Nonisotopic Probes
Authors: Knoll Joan H.M. , Lichter Peter 
Published: June 2004

AbstractNonisotopic in situ hybridization can be used to determine the cellular location and the relative levels of expression of specific transcripts within cells and tissues. RNA in prepared specimens is hybridized with a probe labeled nonisotopically with biotin or digoxigenin, which is generally detected by fluorescence or enzymatic methods. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described in addition to techniques for amplification of weak fluorescent signals obtained in FISH. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described here.
Principles and Application of Fluorescence Microscopy
Authors: Coling Donald , Kachar Bechara 
Published: June 2004

AbstractThis overview discusses the principle of fluorescence along with practical discussions of fluorescent molecular probes, filters and filter sets, multiband filters and multi‐dye fluorescence, light sources, objective lenses, image resolution and the point‐spread function, fluorescence microscopy of living cells, and immunolabeling.
Measurement of In Situ Hybridization
Authors: Hoover Shelley B. , Fox Cecil H. , Bahre Jim , Cottler‐Fox Michele , Sung Cynthia 
Published: June 2004

AbstractHybridization of labeled specific molecular probes to nucleic acids in tissues allows geometric and functional location of gene expression or of foreign genome sequences. Estimates of amounts and location of target nucleic acid sequence can be made with phosphor storage imaging and molecular controls.
Morphological, Biochemical, and Flow Cytometric Assays of Apoptosis
Authors: Martin David , Lenardo Michael 
Published: June 2004

AbstractAs programmed cell death (PCD), or apoptosis, has emerged as an important regulator of development and homeostasis in multicellular organisms, methods to quantify apoptosis and to distinguish it from necrosis have been developed. Necrosis refers to the morphology usually associated with accidental cell death, while apoptosis is seen when cell death is programmed or physiologically regulated. This unit presents a set of assays for these purposes, many of which are technically very simple. Featured in this unit is the TUNEL method of detecting cells that exhibit DNA fragmentation, which can also be performed on tissue sections to locate apoptotic cells in situ.
Enzymatic and Chemical Cleavage of Fusion Proteins
Authors: LaVallie Edward R. , McCoy John M. , Smith Donald B. , Riggs Paul 
Published: June 2004

AbstractThis unit provides protocols for some commonly used methods of site‐specific cleavage of fusion proteins. The first three protocols describe enzymatic cleavage of proteins using proteases (factor Xa, thrombin, and enterokinase) that display highly restricted specificities, which greatly decrease the likelihood that unwanted secondary cuts will occur. Three additional protocols describe specific cleavage of fusion proteins with chemical reagents (cyanogen bromide, hydroxylamine, and low pH) as an alternative to enzymatic cleavage.
Expression and Purification of Maltose‐Binding Protein Fusions
Authors: Riggs Paul 
Published: June 2004

AbstractThis unit describes the procedure for subcloning the sequence encoding the protein of interest into an maltose‐binding protein (MBP) vector, and expressing and purifying the fusion protein from the cytoplasm. MBP vectors include a sequence that encodes the four‐amino‐acid recognition site for the specific protease factor Xa. The site is placed so it can be used to separate the protein of interest from MBP after affinity purification. A support protocol provides a pilot experiment for analyzing the solubility, affinity for the amylose resin, and export of a particular fusion protein. An alternate protocol gives instructions for purifying a fusion protein from the periplasm for fusions that are made in the signal sequence vector and are exported. Additional support protocols detail two different chromatographic methods for separating the protein of interest from MBP after factor Xa cleavage.
Overview of the Baculovirus Expression System
Authors: Murphy Cheryl Isaac , Piwnica‐Worms Helen , Grünwald Stefan , Romanow William G. , Francis Nicole , Fan Hua‐Ying 
Published: June 2004

AbstractBaculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This unit gives an overview of the baculovirus expression system, including discussion of the baculovirus life cycle, and post‐translational modifications that occur in insect cells. In addition, the steps for overproducing proteins in the baculovirus systems are described along with recommendations for choosing an appropriate baculovirus vector and DNA, and reagents and equipment necessary for implementing the whole overexpression system.
Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses
Authors: Murphy Cheryl Isaac , Piwnica‐Worms Helen , Grünwald Stefan , Romanow William G. , Francis Nicole , Fan Hua‐Ying 
Published: June 2004

AbstractThis unit describes the maintenance and care of insect cell cultures as well as the generation, purification, and storage of recombinant baculoviruses. Procedures are included for maintenance and subculturing of insect cells and cotransfection of insect cells with linearized baculovirus DNA and recombinant transfer plasmid containing the gene of interest. In the event that the linearized virus is not available, wild‐type baculovirus (AcMNPV) DNA may be used to produce recombinant baculoviruses. A procedure is also included for the generation of recombinant baculoviruses using a novel method, direct cloning, which eliminates the need to first clone the gene of interest into a baculoviral transfer vector. Preparation of baculovirus infection stocks from both monolayer and suspension cultures is also described. Finally, a protocol is given for a plaque assay to be used for determining the titer of baculoviral stocks as well as for selection of recombinants and plaque purification.
Expression and Purification of Recombinant Proteins Using the Baculovirus System
Authors: Murphy Cheryl Isaac , Piwnica‐Worms Helen , Grünwald Stefan , Romanow William G. , Francis Nicole , Fan Hua‐Ying 
Published: June 2004

AbstractThis unit describes how to analyze protein expression in cells infected with recombinant baculovirus on a small scale for optimizing protein production , how to maximize and scale up recombinant protein production, and how to purify recombinant proteins.
Transient Expression of Proteins Using COS Cells
Authors: Aruffo Alejandro 
Published: June 2004

AbstractThis unit describes the use of COS cells to efficiently produce a desired protein in a short period of time. These cells express high levels of the SV40 large tumor (T) antigen, which is necessary to initiate viral DNA replication at the SV40 origin. Each COS cell transfected with DNA encoding a cell‐surface antigen (in the appropriate vector) or cytoplasmic protein will express several thousand to several hundred thousand copies of the protein 72 hr posttransfection. If the transfected DNA encodes a secreted protein, up to 10 mg of protein can be recovered from the supernatant of the transfected COS cells 1 week posttransfection. COS cell transient expression systems have also been used to screen cDNA libraries, to isolate cDNAs encoding cell‐surface proteins, secreted proteins, and DNA binding proteins, and to test protein expression vectors rapidly prior to the preparation of stable cell lines.
Overview of the Vaccinia Virus Expression System
Authors: Moss Bernard , Earl Patricia L. 
Published: June 2004

AbstractThe vaccinia virus expression system differs from others in that transcription occurs in the cytoplasm of mammalian cells rather than in the nucleus. As a vector, vaccinia virus has a number of useful characteristics, including a capacity that permits cloning large fragments of foreign DNA (20+
kbp) with retention of infectivity, a wide host range, a relatively high level of protein synthesis, and “appropriate” transport, secretion, processing, and posttranslational modifications as dictated by the primary structure of the expressed protein and the cell type used. This overview discusses the life cycle of the vaccinia virus along with effects of vaccinia infection. The vaccinia vector expression system is described along with specific steps for expressing genes using these vectors. Important safety considerations are also presented.
Generation of HIV‐1‐Based Lentiviral Vector Particles
Authors: Ramezani Ali , Hawley Robert G. 
Published: June 2004

AbstractThis unit presents protocols outlining the methodology and techniques involved in the construction and application of HIV‐1‐based lentiviral vector systems. Also described are procedures that can be used to concentrate and purify high‐titer recombinant lentiviral vector preparations, as well as protocols for transduction of adherent and suspension cells.
Direct Chemical Analysis of Glycoconjugates for Carbohydrates
Authors: Manzi Adriana , Esko Jeffrey 
Published: June 2004

AbstractThis unit presents protocols for detection of different sugars bound to glycoconjugates. The solution containing the carbohydrate material is treated with a specific reagent, generating a colored reaction product that can be detected spectrophotometrically. For each assay, the absorbance of the colored solution is proportional to the amount of sugar present in the glycoconjugate.
Detection of Individual Glycosylation Sites on Glycoproteins
Authors: Powell Leland D. 
Published: June 2004

AbstractIn this unit, glycopeptides generated by endopeptidase digestion are first separated by reversed‐phase chromatography. The presence of hydrophilic, negatively charged oligosaccharides shortens retention times, causing glycopeptides to elute in considerably broader peaks than do peptides, so by following the elution profile either radiochemically or colorimetrically, the peaks corresponding to unique glycopeptides can be identified. With proper controls, the number of peaks will correspond to the number of different glycosylation sites. The eluted fractions are suitable for analysis by lectin chromatography, and the peptide sugar linkage can be defined either by endoglycosidase digestion or chemical cleavage. Oligosaccharides freed from the peptide according to the methods described in this unit can be characterized by size or charge, techniques not generally applicable with glycopeptides.
β‐Elimination for Release of O‐GalNAc‐Linked Oligosaccharides from Glycoproteins and Glycopeptides
Authors: Fukuda Minoru 
Published: June 2004

AbstractThis unit describes release of oligosaccharides that are attached to polypeptides through an N‐acetylgalactosamine (GalNAc) linkage to the hydroxyl groups of serine or threonine. The b‐elimination procedures described here can be used to recover the oligosaccharide chains (also called glycans) and/or identify the serine or threonine residues involved in the linkage. A b‐elimination method employing sodium borohydride (NaBH4) and alkaline conditions is described, and an alternative method is also presented in which only alkaline conditions are used without a reducing agent. Another alternative protocol uses sodium sulfite.
Acid Hydrolysis for Release of Monosaccharides
Authors: Manzi Adriana 
Published: June 2004

AbstractThe first step in obtaining a compositional analysis of a glycoconjugate is the release of the individual monosaccharide constituents. Mild acid hydrolysis to release fucosyl residues from glycoconjugates is described in this unit, in addition to mild acid hydrolysis to release sialic acids from glycoconjugates, along with dialysis and column chromatography procedures to purify the sialic acids. Strong acid hydrolysis to release all monosaccharides from glycoconjugates is also detailed.
Enzymatic Release of Oligosaccharides from Glycolipids
Authors: Manzi Adriana E. 
Published: June 2004

AbstractSelective cleavage of the oligosaccharide moiety of glycolipids for further structural analysis can be achieved by means of endoglycoceramidase (EGCase), an enzyme specific for the linkages between oligosaccharide and ceramide residues in glycolipids. The method described here can be used for analysis of glycolipids present in macroscopic amounts or for glycolipids that have been radiolabeled in their carbohydrate moiety.
Endo‐β‐Galactosidases and Keratanase
Authors: Fukuda Michiko N. 
Published: June 2004

AbstractThis overview covers the endo‐β‐galactosidases; enzyme is capable of hydrolyzing a wide range of glycoconjugates. Endo‐β‐galactosidases from numerous sources are discussed in terms of their substrate specificities and substrates, as well as their practical research applications.
Fractionation and Analysis of Neutral Oligosaccharides by HPLC
Authors: Hayes Bradley K. , Varki Ajit 
Published: June 2004

AbstractThis unit describes the fractionation and analysis of neutral oligosaccharides by high‐performance liquid chromatography (HPLC) on bonded amine columns. Separation is based upon hydrogen bonding between the NH2 groups of the column and the hydroxyl groups of the oligosaccharides. A support protocol describes the reduction and desalting of neutral oligosaccharides with sodium borohydride.
Nitrous Acid Degradation of Glycosaminoglycans
Authors: Conrad H. Edward 
Published: June 2004

AbstractGlycosaminoglycans (GAGs) are made up of disaccharide units that are distinguished from each other by the monosaccharide units of which they are composed and by the degree and position of sulfation. These disaccharide units represent the monomeric units of the GAG; thus, measurement of the disaccharide composition of a GAG represents the first step in the characterization of the polymer. In this unit, cleavage of the glycosidic bonds of the N‐sulfated GlcN residues in heparin and heparan sulfate is described, in addition to cleavage of the bonds between the N‐acetylated amino sugar residues in heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate, and hyaluronic acid. Using these procedures involving, all GAGs can be converted completely to their constituent disaccharides and reduced with NaB[3H]4) to yield labeled disaccharides that can be assayed qualitatively or quantitatively. The procedure may also be used to analyze metabolically labeled GAGs (with or without the use of NaB[3H]4).
Analysis of Disaccharides and Tetrasaccharides Released from Glycosaminoglycans
Authors: Conrad H. Edward 
Published: June 2004

AbstractGlycosaminoglycans (GAGs) are converted to disaccharides by various methods. This unit describes separation of individual disaccharides by paper chromatography or paper electrophoresis or high‐performance liquid chromatography (HPLC). Lyase‐released disaccharides can also be monitored by UV absorbance. Support protocols describe mild conditions for reduction of alkali‐labile disaccharides obtained by cleavage of GAGs with lyases, scintillation counting of samples obtained from HPLC separation of radiolabeled saccharides, and calculations for the quantitation of disaccharides.
Analysis of Sulfate Esters by Solvolysis or Hydrolysis
Authors: Freeze Hudson H. 
Published: June 2004

AbstractSulfate esters are found on N‐ and O‐linked sugar chains or glycosaminoglycan (GAG) chains. Few sulfatases are available that can enzymatically remove them, so chemical procedures must be used. These procedures rely on the differential sensitivity of sulfates located in different linkages on the sugar. In comparison to the conditions used for enzymatic digestion, those used for chemical digestion are very harsh and cannot be used on protein‐bound carbohydrates (except for analytical purposes, as described here). With protein‐bound carbohydrates, for preparative purposes, the chains must first be released. This unit describes release of sulfate esters by solvolysis along with a method for monitoring the efficiency of the solvolysis reaction. An alternate procedure provides a scale‐up method for using the solvolysis reaction with large amounts of material. Also presented are techniques for both acid and basic hydrolysis to release sulfate esters.