bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amplification of Cosmid and Plasmid Libraries
Authors: Weis John H. 
Published: June 2004

AbstractThe purpose of amplification is to provide a reagent library that can be used many times. In this protocol, cacteria containing the recombinant clones are grown on agar plates, washed off the plates, and stored in glycerol. An amplified cosmid or plasmid library will contain one library's equivalence in 10 to 100 µl. By producing 100 µl of such a library frozen in 100 1‐ml tubes, over 1000 platings can be expected.
Recombination‐Based Assay (RBA) for Screening Bacteriophage Lambda Libraries
Authors: Kurnit David M. 
Published: June 2004

AbstractThe recombination‐based assay represents a convenient way to screen a complex library constructed in bacteriophage l for homology to a given sequence cloned into a specially designed plasmid. The technique serves to screen a bacteriophage library rapidly and efficiently with a sequence cloned into a plasmid; counterselection then yields the gene product of interest with its plasmid carrier deleted. Because 106 to 107 plaque‐forming units (pfu) may be screened using several petri dishes, and the homology for crossing‐over need only be greater than 25 bp, the RBA represents an efficient way to screen complex l libraries rapidly for homology to a given sequence. In this procedure, a l library is screened using a specially designed plasmid carrying the desired target sequence. Recombinants arising from cross‐over events between the plasmid and a bacteriophage carrying a corresponding region of homology are selected by their ability to grow on strain DM21. Growth of l on DM21 requires the presence of an allele encoded on the plasmid to suppress an amber mutation in the host strain that prevents l propagation. Recovery of the original phage carrying the target sequence requires a reversal of the homologous recombination event. This reversal occurs spontaneously, and is detected by PCR amplification using primers that flank the cloning site in the l vector.
Transfection Using DEAE‐Dextran
Authors: Gulick Tod 
Published: June 2004

AbstractTransfection of cultured mammalian cells using diethylaminoethyl (DEAE)‐dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE‐dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE‐dextran transfection, as well as two more specific protocols for typical experimental applications. The is suitable for transfection of anchorage‐dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE‐dextran‐mediated transfection can be used.
Selection of Transfected Mammalian Cells
Authors: Mortensen Richard , Chesnut Jonathan D. , Hoeffler James P. , Kingston Robert E. 
Published: June 2004

AbstractAnalysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 104 cells in a transfection will stably integrate DNA (the efficiency can vary depending on the cell type). Therefore, a dominant, selectable marker is used to permit isolation of stable transfectants. In the first part of this unit, the procedure for determining selection conditions and the resulting stable transfection is presented and the most commonly used selectable markers are discussed. The second protocol includes conditions for thirteen markers commonly used for selection of mammalian cells. A third protocols describes selection of transfected cells from the total population soon after transfection with plasmids that express both the gene of interest and a selection tag. Optimization of transfection conditions can be facilitated by a simple staining assay detailed in a .
Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics in Vivo
Authors: Kahana Jason A. , Silver Pam A. 
Published: June 2004

AbstractFluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has heretofore been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.
Spectrophotometric and Colorimetric Determination of Protein Concentration
Authors: Simonian Michael H. , Smith John A. 
Published: June 2004

AbstractThis unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measurement at 280 nm is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein. An alternate protocol uses absorbance at 205 nm to calculate the protein concentration. Both methods can be used to quantitate total protein in crude lysates and purified or partially purified protein. Use of a spectrofluorometer or a filter fluorometer to measure the intrinsic fluorescence emission of a sample solution is also described. The measurement is compared with the emissions from standard solutions to determine the concentration of purified protein. The Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to an unknown protein, is also presented, as is the Lowry method, which measures colorimetric reaction of tyrosyl residues in an unknown.
Freezing and Recovery of Hybridoma Cell Lines
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.
Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells
Authors: Abmayr Susan M. , Workman Jerry L. 
Published: June 2004

AbstractExtracts prepared from the isolated nuclei of cultured cells are functional in accurate in vitro transcription and mRNA processing. Thus, such extracts can be used directly for functional studies and as the starting material for purification of the proteins involved in these processes. In this unit, reproducible extracts from different preparations of nuclei are generated. The conditions described are optimized to produce transcriptionally active extracts from HeLa cells. A support protocol describes how to optimize the basic procedure to increase the yield of specific proteins or activities from different cell types, and a second support protocol describes the preparation of the cytoplasmic (S‐100) fraction.
Analysis of DNA‐Protein Interactions Using Proteins Synthesized In Vitro from Cloned Genes
Authors: Struhl Kevin 
Published: June 2004

AbstractTo detect DNA binding activity, radiolabeled protein is incubated with specific DNA fragments, and protein‐DNA complexes are separated from free protein by electrophoresis in native acrylamide gels. Unlike the more conventional mobility shift assay which utilizes
32P‐labeled DNA and unlabeled protein, the assay described here generally utilizes 35S‐labeled protein and unlabeled DNA. Major advantages of this method are that any desired mutant protein can be tested for its DNA‐binding properties simply by altering the DNA template, and the subunit structure (e.g., dimer, tetramer) can be determined.
Yeast Cloning Vectors and Genes
Authors: Lundblad Victoria 
Published: June 2004

AbstractThis unit describes some of the most commonly used yeast vectors, as well as the cloned yeast genes that form the basis for these plasmids. Yeast vectors can be grouped into five general classes, based on their mode of replication in yeast: YIp, YRp, YCp, YEp, and YLp plasmids. With the exception of the YLp plasmids (yeast linear plasmids), all of these plasmids can be maintained in E. coli as well as in S. cerevisiae and thus are referred to as shuttle vectors. The nomenclature of different classes of yeast vectors, as well as details about their mode of replication in yeast are discussed.
In situ Polymerase Chain Reaction and Hybridization to Detect Low‐Abundance Nucleic Acid Targets
Authors: Bagasra Omar , Seshamma Thikkavarapu , Pomerantz Roger , Hanson John 
Published: June 2004

AbstractThis unit presents a novel approach for detecting low‐abundance nucleic acid targets in nuclear and cytoplasmic regions by amplification of specific target sequences using an in situ polymerase chain reaction (ISPCR). If the target sequence is RNA, ISPCR is preceded by in situ reverse transcription. Following ISPCR, in situ hybridization is performed. An describes a variant in situ method for simultaneously reverse transcribing and amplifying RNA transcripts using recombinant Thermos thermophilos (rTth) polymerase. In situ hybridization and detection of amplified targets is also described.
Overview of Image Analysis, Image Importing, and Image Processing using Freeware
Authors: Bearer E.L. 
Published: June 2004

AbstractQuantitative image analysis turns microscopic data that might otherwise be merely descriptive into reliable mechanistic information. This unit provides an overview of the types of analysis that have been useful in the past, as well as descriptions of emerging applications. In addition, two protocols are included for importing data and for the first steps of data manipulation in the more common analytical applications of the freeware, NIH Image and ImageJ, as well as commercially available imaging software, Adobe Photoshop.
Ligation‐Mediated PCR for Genomic Sequencing and Footprinting
Authors: Mueller Paul R. , Wold Barbara , Garrity Paul A. 
Published: June 2004

AbstractThis unit describes how PCR can be used to exponentially amplify segments of DNA located between two specified primer hybridization sites. A single‐sided PCR method is used that initially requires specification of only one primer hybridization site; the second is defined by the ligation‐based addition of a unique DNA linker. This linker, together with the flanking gene‐specific primer, allows exponential amplification of any fragment of DNA. Because a defined, discrete‐length sequence is added to every fragment, complex populations of DNA such as sequence ladders can be amplified intact with retention of single‐base resolution. The ligation‐based protocol was specifically designed for genomic footprinting and direct sequencing reactions, and is described in this context; it can, however, be used for other applications.
Molecular Cloning of PCR Products
Authors: Finney Michael , Nisson Paul E. , Rashtchian Ayoub 
Published: June 2004

AbstractIt is often desirable to clone PCR products to establish a permanent source of cloned DNA for hybridization studies, to obtain high‐quality DNA sequencing results, or to separate products when PCR amplification yields a complex mixture. The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments. This unit describes the strategies for generating and manipulating suitable ends on the PCR fragments.
Expression and Purification of Thioredoxin Fusion Proteins
Authors: McCoy John , LaVallie Edward 
Published: June 2004

AbstractThis unit describes a gene fusion expression system that uses thioredoxin, the product of the Escherichia coli trxA gene, as the fusion partner. The system is particularly useful for high‐level production of soluble fusion proteins in the E. coli cytoplasm; in many cases heterologous proteins produced as thioredoxin fusion proteins are correctly folded and display full biological activity. Protein fusions to His‐patch Trx can usually be purified in a single step from cell lysates. Additional protocols describe E. coli cell lysis using a French pressure cell and fractionation, osmotic release of thioredoxin fusion proteins from the E. coli cytoplasm, and heat treatment to purify some thioredoxin fusion proteins.
Expression and Purification of Epitope‐Tagged Multisubunit Protein Complexes from Mammalian Cells
Authors: Wu Shwu‐Yuan , Chiang Cheng‐Ming 
Published: June 2004

AbstractBiochemical characterization and functional studies of mammalian proteins are often hampered by the availability of the purified protein, in particular, when the functional entity is present as a multisubunit protein complex in the cell. To overcome the difficulties in the purification of multisubunit protein complexes from mammalian cells, one may create stable cell lines containing epitope‐tagged protein. The first protocol in this unit describes the procedures involved in the establishment of a stable cell line constitutively expressing the FLAG‐tagged protein by retrovirus‐mediated gene transfer and immunoaffinity purification of the epitope‐tagged multisubunit protein complex. The next protocol outlines the steps involved in the establishment of an inducible cell line conditionally expressing the FLAG‐tagged protein by a tetracycline‐regulated system, and the one‐step immunoaffinity purification of the multisubunit protein complex. An alternate protocol provides an excellent example for the purification of different forms of human RNA polymerase II complexes, achieved simply by choosing the appropriate starting material and by varying wash conditions. The isolation of various human TATA‐binding protein (TBP)‐containing complexes is described in a support protocol, and is a good example of combining the P11 column and immunoaffinity purification. These protocols, collectively, illustrate a powerful methodology in applying epitope tagging and stable cell line approaches for the purification of multisubunit protein complexes from mammalian cells.
Metabolic Radiolabeling of Animal Cell Glycoconjugates
Authors: Diaz Sandra , Varki Ajit 
Published: June 2004

AbstractThis unit describes metabolic labeling techniques that provide specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. Although these techniques provide less information than complete sequencing of the sugar chains, the partial structural information derived is sufficient for many purposes. In the basic procedure presented in this unit, actively growing cell cultures are grown through several population doublings in complete medium supplemented with radiolabeled glycoconjugate precursors to reach a steady‐state level of incorporation. In alternate protocols, cells are cultured for a short period of time in a deficient medium that contains a high concentration of radiolabeled precursor. A pulse or pulse‐chase labeling procedure can be used to analyze precursor‐product relationships. With sequential pulse‐labeling, it is possible to obtain quantities of labeled glycoconjugates with the use of a minimal amount of labeled precursor by using the same medium to pulse‐label a series of cultures. A support protocol describes the preparation of multiply deficient medium (MDM) for use in making appropriate deficient media.
Inhibition of N‐Linked Glycosylation
Authors: Powell Leland D. 
Published: June 2004

AbstractTreatment of cells with inhibitors of the enzymes that synthesize N‐linked oligosaccharide chains results in production of glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of this class of oligosaccharides on specific proteins or intact cells. This unit describes the use of inhibitors to prevent N‐linked glycosylation of proteins in cultured cells. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring [35S]methionine incorporation as a measure of protein biosynthesis. The inhibitor's ability to inhibit oligosaccharide processing is then determined by analyzing cells labeled with [HH]mannose using TCA precipitation or endo H digestion (UNIT 13). Further suggestions are given on how to use methods for identifying a specific glycoprotein (if available) to measure the effect of the inhibitor on its N‐linked oligosaccharide chains. A support protocol details a method for concentrating proteins by acetone precipitation.
Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis and Inhibitors of Glycoprotein and Proteoglycan Assembly
Authors: Esko Jeffrey D. , Montgomery Rebecca I. 
Published: June 2004

AbstractWith the exception of hyaluronic acid, all mammalian saccharides assemble while attached to a lipid or protein primer. Several cases are now known in which oligosaccharide synthesis will occur on synthetic glycoside primers added to cells. A protocol is described in this unit in which b‐D‐xylosides initiate glycosaminoglycan (GAG) synthesis by substituting for endogenous xylosylated core proteins. At high concentration xylosides will also prime oligosaccharides that resemble glycolipids. N‐acetyl‐a‐D‐galactosaminides initiate the synthesis of O‐linked oligosaccharides found on mucins and other glycoproteins in an analogous manner. Even disaccharides, such as peracetylated N‐acetyllactosaminide, can act as primers. Because these primers compete with endogenous substrates, they also act as inhibitors of proteoglycan (PG) and glycoprotein synthesis. Thus, primers have utility for studying the biological activity of glycoconjugates in cells, tissues, and animals. This unit describes procedures for using glycoside primers in cell culture.
Total Compositional Analysis by High‐Performance Liquid Chromatography or Gas‐Liquid Chromatography
Authors: Manzi Adriana E. 
Published: June 2004

AbstractOnce the presence of carbohydrate in a glycoprotein has been confirmed, the next step is to determine the precise molar ratio of its monosaccharide constituents. The analysis involves two major phases. The release of the individual monosaccharides is achieved by methanolysis (described here), total acid hydrolysis, or enzymatic release of sialic acids. The resulting mixtures of monosaccharides are then analyzed by methods described in this unit: fractionation, characterization, and quantitation by high‐performance liquid chromatography using anion‐exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) and other HPLC systems or by gas‐liquid chromatography with flame ionization detection. The identity of the individual monosaccharides is determined by comparison with known standards processed and analyzed in the same way.
Internet Basics for Biologists
Authors: Baxevanis Andreas D. , Ouellette B.F. Francis 
Published: June 2004

AbstractBefore embarking on any practical discussion of computational methods in solving biological problems, it is necessary to lay the common groundwork that will enable users to both access and implement the algorithms and tools discussed in this book. This unit begins with a review of the Internet and its terminology, and also discusses major classes of Internet protocols, without becoming overly engaged in the engineering minutiae underlying these protocols. This unit also discusses matters of connectivity, ranging from simple modem connections to digital subscriber lines (DSL). Finally, one of the most common problems that has arisen with the proliferation of Web pages throughout the world is addressed, i.e., finding useful information on the World Wide Web.
Affinity Purification of Proteins Binding to GST Fusion Proteins
Authors: Swaffield Jonathan C. , Johnston Stephen Albert 
Published: June 2004

AbstractThis unit describes the use of proteins fused to glutathione‐S‐transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose‐binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding.
Two‐Hybrid Dual Bait System
Authors: Kotova Elena , Serebriiskii Ilya , Coleman Thomas 
Published: June 2004

AbstractThe yeast two‐hybrid system, or interaction trap, is one of the most versatile methods available with which to identify and establish protein‐protein interactions. The dual bait system is one adaptation of the classic approach. This system facilitates the simultaneous comparison of two distinct baits with one prey. One protein of interest is expressed as a fusion to the DNA binding protein LexA (bait 1), while a second protein of interest is expressed as a fusion to the DNA binding protein cI (bait 2). Strains of yeast engineered for screening of these dual baits possess four separate reporter genes. A plasmid expressing an activation domain‐fused protein (prey), which can be either a defined protein interactor or a cDNA library, is also expressed to allow dual hybrid‐mediated transcriptional activation.
Gene Silencing by RNAi in Mammalian Cells
Authors: John Matthias , Geick Anke , Hadwiger Philipp , Vornlocher Hans‐Peter , Heidenreich Olaf 
Published: June 2004

AbstractThis unit provides information how to use short interfering RNA (siRNA) for sequence specific gene silencing in mammalian cells. Several ways for siRNA generation and optimisation, as well as recommendations for cell transfection are presented.
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Semba Kentaro 
Published: June 2004

AbstractThis appendix presents useful basic information, including common abbreviations, useful measurements and data, characteristics of amino acids and nucleic acids, information on radioactivity and the safe use of radioisotopes and other hazardous chemicals, conversions for centrifuges and rotors, characteristics of common detergents, and common conversion factors.
Removal of Lymphoid Organs
Authors: Reeves J.P. , Reeves P.A. 
Published: June 2004

AbstractAlthough for most experiments lymphoid tissue is removed from the freshly sacrificed animal, there are several circumstances which require the surgical removal, under anesthesia, of either the thymus or spleen, and the recovery of the animal for further investigation. This unit describes survival surgery for removal of the adult spleen as well as the adult and neonatal thymus of the mouse.