bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lectin Analysis of Proteins Blotted onto Filters
Authors: Freeze Hudson H. 
Published: June 2004

AbstractLectins are proteins that bind with great specificity to certain carbohydrate structures. Plant lectins are widely used for investigations of carbohydrate structure and for fractionation and purification of individual oligosaccharides and glycopeptides. This unit describes the use of lectins as sensitive indicators for the presence of certain carbohydrate structures linked to proteins blotted onto filters. A tagged lectin is incubated with a blot containing the target protein and binding of the lectin is detected by one of several different procedures. Direct approaches include using lectins labeled with 125I or conjugated to horseradish peroxidase or alkaline phosphatase, which can be detected by chromogenic or luminescent visualization systems. Indirect approaches involve using lectins conjugated to biotin or digoxigenin followed by a second incubation with alkaline phosphatase‐conjugated avidin or antibodies specific for the haptenic digoxigenin group and then by visualization. Several commercial kits are available that provide labeled lectins, control proteins, and developing reagents needed for visualization. These systems can also be adapted for use with lectins other than those supplied with kits. The protocol in this unit is easy to perform with or without a kit. However, the results, while suggestive of carbohydrate structure, are not definitive.
Detection of Glycophospholipid Anchors on Proteins
Authors: Doering Tamara L. , Englund Paul T. , Hart Gerald W. 
Published: June 2004

AbstractMany eukaryotic proteins are tethered to the plasma membrane by glycosyl phosphatidylinositol (GPI) membrane anchors. This unit provides a general approach for detecting GPI‐anchored proteins. First, the detergent‐partitioning behavior of a protein of interest is examined for characteristics of GPI‐linked species. The protein may also be subjected to specific enzymatic or chemical cleavages to release the protein from its GPI anchor. Protocols for phospholipase cleavage and chemical cleavage with nitrous acid are provided for this purpose. If GPI‐anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. Separation of lipid moieties and base hydrolysis of proteins are detailed herein.
Inhibition of Glycolipid Biosynthesis
Authors: Manzi Adriana E. 
Published: June 2004

AbstractAdequate inhibition of glycolipid biosynthesis allows the study of their biological functions. The method presented in this unit employs a synthetic analog of ceramide, PDMP (1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol), that inhibits glycolipid biosynthesis in cultured cells. Optimum conditions for inhibition of glycolipid biosynthesis are determined, glycolipids extracted from cultures grown with and without inhibitor, and the patterns of glycolipids analyzed by HPTLC. Detection is achieved using colorimetric reactions, or by monitoring radioactivity when cells have been metabolically radiolabeled.
β‐Elimination for Release of O‐Linked Glycosaminoglycans from Proteoglycans
Authors: Conrad H. Edward 
Published: June 2004

AbstractO‐linked glycosaminoglycan (GAG) chains in proteoglycans are readily released from their core proteins by treatment with alkali at room temperature. This β‐elimination is the same type of reaction as that for releasing O‐linked oligosaccharides from their core proteins. Under the reaction conditions described here, N‐linked oligosaccharides remain attached to the core protein, but any O‐linked oligosaccharides will be released along with the GAG chains. The procedure can be used to isolate the free GAG chains, the free O‐linked oligosaccharides, and the core protein (which will still have any N‐linked oligosaccharides that were originally present).
Analysis of Monosaccharides
Authors: Manzi Adriana E. , Powell Leland D. , Varki Ajit 
Published: June 2004

AbstractThis unit presents methods for assaying sialic acids, reducing sugars, and hexosamines. The BCA assay detects free reducing terminii in sugars released from glycoconjugates by appropriate treatments. Assays employing Ehrlich reagent (DMAB) detect hexosamines and N‐acetylhexosamines, including a method for hydrolyzing the glycosidic linkages of the hexosamines and a method for re‐N‐acetylation. The TBA and DMB assays can be used to quantitate and fractionate free forms of many types of sialic acids. Techniques for liberating the sialic acids from the parent glycoconjugates are also provided.
Analysis of Oligosaccharide Negative Charge by Anion‐Exchange Chromatography
Authors: Varki Ajit 
Published: June 2004

AbstractThis unit presents the analysis of negative charge on labeled N‐ or O‐linked oligosaccharides. These protocols may be used in the initial screening of oligosaccharides to detect negative charge, for analytical or preparative separation of oligosaccharides based on their negative charge, or to analyze the type of negative charge found on the oligos accharides. The describes the use of anion‐exchange (QAE‐Sephadex) chromatography with stepwise elution for estimating the number of negative charges on an oligosaccharide sample derived from glycosidase treatment of a glycoprotein. In an , gradient elution is used for the preparative separation of oligosaccharides based on negative charge. A describes a method for measuring loss of or change in negative charge after treatment of the oligosaccharide sample with mild acid and/or phosphatases.
HPLC Methods for the Fractionation and Analysis of Negatively Charged Oligosaccharides and Gangliosides
Authors: Manzi Adriana E. , Hayes Bradley K. 
Published: June 2004

AbstractThis unit describes the fractionation and analysis of anionic oligosaccharides and gangliosides using anion‐exchange high‐performance liquid chromatography (HPLC). Saccharides or gangliosides are eluted in order of the number of negative charges they possess, although the charge‐to‐mass ratio can also contribute to elution position.
Overview of Protein Phosphorylation
Authors: Sefton Bartholomew M. , Shenolikar Shirish 
Published: June 2004

AbstractThis overview discusses the significance and roles of protein phosphorylation in regulation of protein function. Sites of phosphorylation are described as well as methods for detecting both radiolabeled and unlabeled phosphoamino acids. Importantly, protein kinases and phosphatases, the regulators of phosphorylation are discussed.
Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
Authors: Sefton Bartholomew M. 
Published: June 2004

AbstractThis unit describes 32Pi labeling and lysis of cultured cells to be used for subsequent immunoprecipitation of proteins. The approach is appropriate, however, for labeling any cellular constituent with 32P. This procedure is suitable for insect, avian, and mammalian cells and can be used with both adherent and nonadherent cultures. The general approach involves biosynthetic labeling with 32Pi in medium containing a reduced concentration of phosphate. This approach can also be modified to label any cellular constituents with 32Pi. The first procedure described is 32Pi labeling of adherent or nonadherent (e.g., hematopoietic) cells with subsequent lysis in a detergent buffer. More rigorous lysis conditions to be used for working with proteins that are difficult to solubilize are also described.
Phosphoamino Acid Analysis
Authors: Sefton Bartholomew M. 
Published: June 2004

AbstractIt is often valuable to identify the phosphorylated residue in a protein. In the case of proteins phosphorylated at serine, threonine, or tyrosine, this is readily accomplished by partial acid hydrolysis in HCl followed by two‐dimensional thin‐layer electrophoresis of the labeled phosphoamino acid, as described here. Phosphothreonine andphosphotyrosine are more stable to hydrolysis in alkali than are RNA andpho sphoserine. Therefore, a protocol for mild alkaline hydrolysis of protein samples is also provided to enhance the detection of phosphothreonine and phosphotyrosine. Although this procedure can be carried out with a protein eluted from a preparative gel and concentrated by trichloroacetic acid or acetone precipitation, it is most easily accomplished by transfer of the protein of interest to a PVDF membrane.
Detection of Phosphorylation by Enzymatic Techniques
Authors: Shenolikar Shirish 
Published: June 2004

AbstractReversible protein phosphorylation is an important mechanism for regulating physiological processes in both plant and animal cells. There are a number of techniques to demonstrate the presence of covalently bound phosphate in proteins. The general strategy of the protocols in this unit is to first examine the functional effects elicited by nonspecific acid or alkaline phosphatases that dephosphorylate many phosphoproteins in vitro. Protein phosphatases that selectively hydrolyze phosphoserine and phosphothreonine or phosphotyrosine residues can then be used to identify a functionally important covalent modification. Additional protocols describe digestion of phosphoproteins with a protein serine/threonine phosphatase and protein tyrosine phosphatase. A support protocol has been included to identify the radiolabel as 32Pi based on its ability to form a complex with ammonium molybdate.
Assays of Protein Kinases Using Exogenous Substrates
Authors: Carter A. Nigel 
Published: June 2004

AbstractIn studies of the regulation of specific biochemical events by reversible phosphorylation, assaying the protein kinases themselves can often lead to significant progress in understanding the mechanistic details of a system under study. This unit describes assays for a variety of protein kinases that require different conditions to detect and measure their activities: cyclic nucleotide‐dependent kinases, protein kinase C and isoforms, casein kinases, Ca2+/calmodulin‐dependent kinases, and tyrosine kinase. A protocol for in‐gel assays for specific kinase activity is also provided. The unit is not meant to be a catalog of individual protein kinase assays; however, the general principles of these assays should apply to most if not all known protein kinases.
Sequence Databases: Integrated Information Retrieval and Data Submission
Authors: Weisemann Jane M. , Boguski Mark S. , Ouellette B.F. Francis 
Published: June 2004

AbstractThis unit provides an overview of biomedical information resources, focusing on sequence data, structure information, and the associated literature, and also discusses how nucleotide sequence data gets into the databases in the first place. Some specific databases covered here are MEDLINE, GenBank, and Entrez.
Protein Databases on the Internet
Authors: Xu Dong , Xu Ying 
Published: June 2004

AbstractThis unit provides a starting point for readers to explore the potential of protein databases on the Internet. Databases for different aspects of proteins are discussed, with the focus on sequence, structure, and family. The strengths and weaknesses of the databases are addressed. From hundreds of on‐line protein databases, several major databases are discussed as examples to illustrate their features and how they can be used effectively. Most other protein databases can be explored in a similar way.
Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins
Authors: Golemis Erica A. , Serebriiskii Ilya , Finley Russell L. , Kolonin (hunt by interaction mating) Mikhail G. , Gyuris Jeno , Brent Roger 
Published: June 2004

AbstractThe parameters and experimental conditions for this important system are constantly undergoing improvement. This newest version includes expanded tables describing interaction trap components and additional libraries compatible with the interaction trap system. It also features a new protocol on performing a hunt by interaction mating. Some of the commercial vendors selling yeast two‐hybrid reagents recommend using interaction mating to perform a hunt, so this procedure should be of great interest.
Surface Plasmon Resonance for Measurements of Biological Interest
Authors: Bamdad Cynthia 
Published: June 2004

AbstractGenetic manipulations, including gene knock‐outs and mutant screens, provide an initial hint as to the function of a gene product and indicate possible associated factors. To unravel complicated biological processes, which control the development of organisms, one must identify the interacting components. An in vitro technique based on an optical phenomenon, called surface plasmon resonance (SPR), can simultaneously detect interactions between unmodified proteins and directly measure kinetic parameters of the interaction. This technique is gaining popularity, due to the increased availabilty oif user‐friendly machines, and an overview of the technology is presented in this unit.
Interaction Trap/Two‐Hybrid System to Identify Loss‐of‐Interaction Mutant Proteins
Authors: Mendelsohn Andrew R. 
Published: June 2004

AbstractProtein‐protein interactions play a critical role in biology. Disrupting a specific protein‐protein interaction and studying the resulting phenotype can help elucidate the function of the interaction. A method to rapidly make and identify mutant proteins that no longer bind to a specific partner protein is described. The method takes advantage of the ease of the interaction trap/two‐hybrid system and PCR mutagenesis. PCR fusion of the target protein to GFP is used to ensure the protein's open reading frame is not disrupted by mutagenesis. The resulting noninteracting mutant proteins usually result from a single missense mutation, which can easily be identified.
Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific Genomic Sequences In Vivo
Authors: Aparicio Oscar , Geisberg Joseph V. , Struhl Kevin 
Published: June 2004

AbstractChromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein‐protein and protein‐ DNA cross‐links between molecules in close proximity on the chromatin template in vivo. DNA sequences that cross‐link with a given protein are selectively enriched and reversal of the formaldehyde cross‐link permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein‐protein and protein‐ DNA interactions at a particular time point, and hence is useful for kinetic analysis of events occurring on chromosomal sequences in vivo. In addition, ChIP can be combined with microarray technology to identify the location of specific proteins on a genome‐wide basis. This unit describes the ChIP protocol for Saccharomyces cerevisiae; however, it is also applicable to other organisms.
Isolation of Histones and Nucleosome Cores from Mammalian Cells
Authors: Schnitzler Gavin R. 
Published: June 2004

AbstractIn vitro analysis of DNA in chromatin is often important for understanding mechanisms of regulation of transcription and other processes that occur on DNA. The basic unit of chromatin is the nucleosome core, containing two copies each of the core histones H2A, H2B, H3, and H4 to form a histone octamer that wraps 145 base pairs of DNA in a left‐handed superhelix. In vivo, chromatin is associated with linker histones (such as H1), which facilitate the ordered packing of nucleosomes. This linker histone‐containing particle is properly termed the nucleosome (or chromatosome), while the linker histone‐free particle is the nucleosome core. Pure polynucleosome cores and histones can be readily isolated from mammalian tissue culture cells. This unit describes procedures for isolation and purification of nuclei, isolation of polynucleosomes lacking linker histones from these nuclei, isolation of pure populations of mono‐ and dinucleosome cores from oligonucleosome fractions, and isolation of core histones from purified nuclei. The methods presented here do not denature the histones, and may yield histones that are more active for in vitro assemblies.
Profiling Human Gene Expression with cDNA Microarrays
Authors: Jiang Yuan , Lueders John , Glatfelter Arthur , Gooden Chris , Bittner Michael 
Published: June 2004

AbstractAs a result of genomics initiatives worldwide, it has become increasingly easy to obtain cDNA clones representing the 3' ends of many human genes. This unit describes methods that allow these clones to be used as hybridization detectors in a highly parallel assay of gene expression. Protocols are provided for preparing cDNA microarrays, extracting RNA from cells of interest and preparing fluorescently labeled cDNA representations of the message pools, and hybridizing the labeled cDNAs to the microarrays.
Mouse Embryo Fibroblast (MEF) Feeder Cell Preparation
Authors: Conner David A. 
Published: June 2004

AbstractMitotically inactive mouse embryo fibroblasts (MEFs) are commonly used as feeder layers to prevent the differentiation of mouse embryonic stem (ES) cells. This unit describes the isolation of MEFs and the use of g‐irradiation or mitomycin C to produce inactivated feeders suitable for the culture of ES cells.
Mouse Embryonic Stem (ES) Cell Culture
Authors: Conner David A. 
Published: June 2004

AbstractMouse embryonic stem (ES) cells are used to generate mouse mutants by gene targeting and blastocyst‐mediated transgenesis. ES cells must be cultured under conditions that prevent differentiation to maintain their ability to transmit altered alleles by contributing to the germ line. This unit describes one of the most common methods for culturing ES cells using mouse embryo fibroblast (MEF) feeder layers and recombinant leukemia inhibitory factor (LIF) to prevent differentiation.
Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)
Authors: Mortensen Richard 
Published: June 2004

AbstractFormerly UNIT 9.16, this unit takes a more appropriate place in Chapter 23, and has been updated and revised for this publication. Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. In this unit, the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker (e.g., neo). Expression of neo is obtained by including the phosphoglycerate kinase (PGK) promoter in the construct. To enrich for clones in which the target gene has undergone homologous recombination over those in which random integration of the construct has occurred, a negative selectable marker, herpes simplex virus thymidine kinase (HSV‐TK), is included in the construct outside the region of homology to the target gene. Depending upon the target gene, it may be easier to assemble the construct by adding the neo and TK genes to the cloned target gene or by adding two fragments of the target gene to a plasmid containing the neo and TK genes.
Design, Synthesis, and Amplification of DNA Pools for Construction of Combinatorial Pools and Libraries
Authors: Pollard Jack , Bell Sabine D. , Ellington Andrew D. 
Published: June 2004

AbstractThis unit describes the design, synthesis, and amplification of random‐sequence DNA pools, from which functional nucleic acid‐binding or catalytic species can be selected. Since it is an expensive and time‐consuming process, the authors have provided an extensive strategic planning section to guide investigators in designing and constructing the pool.
Preparation of Single Cells from Solid Tissues for Analysis by PCR
Authors: Sawtell N.M. 
Published: June 2004

AbstractThis unit details a protocol for the separation of solid tissues into single cell suspensions for subsequent analysis of nucleic acids and protein.Tissues are fixed in situ by perfusion, which terminates cell processes and thus changes that would accompany dissociating the living tissue. The procedure is particularly useful when the cell type of interest represents a minor population relative to other cells types in the tissue. Once separated, individual cells or groups of a particular cell type can then be analyzed using PCR strategies. The procedure can also be adapted to allow the quantification of the number of cells within a tissue containing specific nucleic acid sequences, for example, a particular viral DNA or RNA sequence.
PCR‐Based Subtractive cDNA Cloning
Authors: Patel Mukesh , Sive Hazel 
Published: June 2004

AbstractSubtractive cloning is a powerful technique that allows isolation and cloning of mRNAs differentially expressed in two cell populations. In the generalized subtraction scheme the cell types to be compared are the [+] or tracer cells and the [‐] or driver cells, where mRNAs expressed in the tracer and not the driver are isolated. Briefly, tracer nucleic acid (cDNA or mRNA) from one cell population is allowed to hybridize with an excess of complementary driver nucleic acid from a second cell population to ensure that a high percentage of the tracer forms hybrids. Hybrids that form include sequences common to both cell populations. Hybrids between the tracer and driver, and all driver sequences, are removed in the subtraction step. The unhybridized fraction is enriched for sequences that are preferentially expressed in the tracer cell population. The method described in this unit uses double‐stranded cDNA (ds cDNA) as both tracer and driver. Reciprocal subtractions are performed between two cell populations, A and B:
that is, genes preferentially expressed in A more than in B are isolated, as are genes expressed preferentially in B more than in A. The method uses the polymerase chain reaction (PCR) to amplify cDNAs after each subtraction to prepare tracer and driver for the next subtraction. The progress of subtraction is monitored by slot blot hybridization. Differentially expressed cDNA sequences are used to construct a subtracted cDNA library.
Overview of RNA Interference and Related Processes
Authors: Baulcombe David 
Published: June 2004

AbstractThe history of RNA interference (RNAi) has unfolded rapidly since 1997 with a series of discoveries from plants, fungi, and animals. Initially, the interest was directed towards development of gene silencing technology that could be applied in research, medical therapy, and crop improvement. However, as the underlying mechanism was revealed, it became apparent that RNAi manifests a novel system of genetic regulation that was only hinted at by previous data. This overview unit gives a brief history of the field, describes the natural role of RNAi and related processes, identifies strategies for RNAi and cosuppression in plants and animals, and describes methods for detection and characterization of siRNA and miRNA.
Cloning of Small RNA Molecules
Authors: Pfeffer Sébastien , Lagos‐Quintana Mariana , Tuschl Thomas 
Published: June 2004

AbstractSmall RNAs that are derived from double‐stranded RNA precursors act as guide RNAs during sequence‐specific epigenetic regulation of eukaryotic gene expression. These small regulatory RNAs are between 20 and 30 nucleotides in length, and fall into one or more of the following categories: small interfering RNAs (siRNAs), microRNAs (miRNAs), and heterochromatic siRNAs (hsiRNAs). Procedures to record the profile of small RNAs expressed in cultured cells or tissues are described. The small RNAs are directionally cloned after isolation from total RNA. The methods rely on T4 RNA ligase‐based joining of adapter oligonucleotides to the 3( and 5( termini of the pool of small RNAs. The ligation products are reverse transcribed and PCR‐amplified. It is recommended to directionally concatamerize the relatively short PCR products before cloning in order to increase the number of RNA sequences obtained per clone.