bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of Glutathione‐Related Enzymes
Authors: Esworthy R.S. , Chu F.‐F. , Doroshow J.H. 
Published: June 2004

AbstractGlutathione peroxidase activity can be measured using a variety of substrates to define total glutathione peroxidase activity or the activity of subsets of the family of enzymes. The assays for these activities are spectrophotometric assays, and the specificity depends on the choice of substrate and/or prior treatment of the samples by chromatography or immunoprecipitation. This unit provides the assays and support protocols for synthesizing substrates and calculating specific activity.
Overview of Heme Degradation Pathway
Authors: Maines Mahin D. 
Published: June 2004

AbstractHeme oxygenase 1 and 2 activities are responsible for initiating most of the degradation of heme, although other enzyme pathways play a role as well. The degradation pathway also includes biliverdin reductase, the activity of which is coupled to oxidation of NADH and NADPH. This overview discusses the pathways and enzymes involved in heme degradation.
Detection of Heme Oxygenase 1 and 2 Proteins and Bilirubin Formation
Authors: McCoubrey William K. 
Published: June 2004

AbstractIt is possible to detect changes in the level of expression of the two isozymes of heme oxygenase (HO). The sequence differences allow the production of isozyme‐specific antibodies that can be used in immunoblot analysis. Total HO activity in tissue microsomal fractions or extracts of bacteria transformed with an HO gene can be measured as described in this unit using a coupled assay employing biliverdin reductase, which converts the biliverdin product to bilirubin. Bilirubin is then quantified with a scanning spectrophotometer.
An HPLC Method to Detect Heme Oxygenase Activity
Authors: Ryter Stefan W. , Tyrrell Rex M. 
Published: June 2004

AbstractThis unit presents a method to calculate heme oxygenase enzymatic activity from the formation of bilirubin equivalents [biliverdin‐Ixalpha (BV) and bilirubin‐IXalpha (BR)]. The BV and BR generated in the reaction are separated by reversed‐phase HPLC and detected using visible absorbance spectroscopy. Since both metabolites of heme degradation are directly quantifiable, the assay eliminates the requirement for biliverdin reductase supplementation.
Measurement of cGMP and Soluble Guanylyl Cyclase Activity
Authors: Southam Eric 
Published: June 2004

AbstractSoluble guanylyl cyclase is responsible for synthesis of guanosine 3'5'‐cyclic monophosphate. The enzyme is activated by NO, so measurement of tissue levels of cGMP and the activity of guanylyl cyclase is important in studies of NO‐cGMP signal transduction. The method of choice for measuring tissue levels of cGMP is a radioimmunoassay, but it can also be measured using a scintillation proximity assay. Guanylyl cyclase activity is determined by measuring the conversion of GTP to cGMP using a radioimmunoassay or by following the conversion of [32P]GTP to [32P]cGMP, with separation of the product by precipitation and chromatography on an alumina column.
Principles of Electrophysiology: An Overview
Authors: Narahashi Toshio 
Published: June 2004

AbstractThis overview gives brief accounts of the mechanism of nerve excitation, receptor/channel functions, cable properties of neurons, and voltage clamp and patch clamp principles. Among various aspects of electrophysiology, emphasis is placed on the mechanistic studies of receptors/channels. A few sample records of membrane currents are illustrated to aid reader's comprehension. Finally some useful reference books are given for more comprehensive information.
Electrophysiological Studies of Neurotoxicants on Central Synaptic Transmission in Acutely Isolated Brain Slices
Authors: Yuan Yukun , Atchison William D. 
Published: June 2004

AbstractEffects of neurotoxic chemicals on central synaptic function can be assessed using electrophysiological recording techniques in freshly isolated brain slices. The three most commonly used electrophysiological recording methods in brain slices are extracellular microelectrode recording, intracellular microelectrode recording, and whole‐cell patch clamp recording. This unit presents the basic procedures and applications of extra‐ and intracellular recordings in hippocampal slices and whole‐cell recording in cerebellar slices.
Isolation of Liver Kupffer Cells
Authors: Froh Matthias , Konno Akira , Thurman Ronald G. 
Published: June 2004

AbstractIsolation of Liver Kupffer Cells (Matthias Froh, Akira Konno, Ronald G. Thurman, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina). Kupffer cells, the resident macrophages of the liver, play a major role in the pathogenesis of several diseases. This unit contains an easy‐to‐follow procedure for effective isolation of liver Kupffer cells from rats and mice. The protocol provides viable Kupffer cells in large amounts that can be used for further investigations.
Embryonic Stem (ES) Cell Culture Basics
Authors: Schmidt Jennifer V. 
Published: June 2004

AbstractThis unit describes the procedures to grow and maintain embryonic stem (ES) cells in culture. Growth‐inactivated mouse embryo fibroblasts (MEF) are required as a feeder layer for the ES cells; their isolation and culture is described as are the additional protocols required for generating a mouse with a particular targeted gene electroporation, cloning and selection for homologous recombinants, transfection with Cre, and preparation of cells for injection.
Overview of the Physical State of Proteins within Cells
Authors: Petty Howard R. 
Published: June 2004

AbstractTo understand how proteins work it is necessary to understand their physical state within the cell. This unit reviews the classification of proteins, how that is related to the hydropathicity of the protein, other factors that affect the heterogeneity of proteins, protein assemblies, methods for altering the solubility of proteins, and limitations of in vitro manipulations of proteins.
Computational Methods for Protein Sequence Analysis
Authors: Michaels George , Garian Robert 
Published: June 2004

AbstractThis unit is presented as a guide to addressing the issue of what to do with a protein sequence once it is obtained. A theoretical background for protein sequence analysis is provided first, followed by a discussion of matrix methods for sequence comparison (). Sequence similarity searching is then presented, including the BLAST and FASTA databases. Other aspects of protein sequence analysis covered here are alignment methods, scoring matrices, multiple alignments, cluster methods and trees, and identification of functional sites.
Internet Basics
Authors: Baxevanis Andreas D. , Ouellette B.F. Francis 
Published: June 2004

AbstractWith the explosion of sequence and structural information available to researchers, the field of bioinformatics is playing an increasingly large role in the study of fundamental biomedical problems. The challenge facing computational biologists will be to aid in gene discovery and in the design of molecular modeling, site‐directed mutagenesis, and experiments of other types that can potentially reveal previously unknown relationships with respect to the structure and function of genes and proteins. This challenge becomes particularly daunting in light of the vast amount of data that has been produced by the Human Genome Project and other systematic sequencing efforts to date. This unit begins with a review of the Internet and its terminology, also discussing major classes of Internet protocols, without becoming overly engaged in the engineering minutiae underlying these protocols. Matters of connectivity, ranging from simple modem connections to digital subscriber lines (DSL) are also discussed. 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.
Protein Tertiary Structure Modeling
Authors: Guex Nicolas , Schwede Torsten , Peitsch Manuel C. 
Published: June 2004

AbstractInsights into the 3D‐structure of a protein have proven useful during experiment design. Experimentally elucidated structures are often not available, but comparative protein modeling provides a viable alternative in many cases. This unit presents comparative protein modeling and how to use the highly sophisticated but easy‐to‐use free software available on the Internet.
Overview of Protein Expression in Pichia pastoris
Authors: Higgins David R. 
Published: June 2004

AbstractPichia pastoris is a methylotrophic yeast and can be used as a heterologous expression system. This microorganism is as easy to manipulate as Escherichia coli, but has many of the advantages of eukaryotic expression (e.g., protein processing, folding, and post‐translational modifications), and it is faster, easier, and cheaper to use than other eukaryotic expression systems, such as baculovirus or mammalian tissue culture. It also generally yields higher expression levels. This overview discusses important considerations for the use of Pichia pastoris, including strains for expression, expression plasmids, transformation by integration, and post‐translational modifications. Examples of expression are given and finally, legal issues regarding patent rights for heterologous protein expression in Pichia pastoris are described.
Production of Recombinant Proteins in Mammalian Cells
Authors: Chen Su , Gray David , Ma Johnny , Subramanian Shyamsundar 
Published: June 2004

AbstractThe best strategy for consistent production of larger quantities of pure protein is stable expression. Popular hosts for stable expression are Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK‐21) cells, myeloma cells, and the transformed kidney cell line 293. Protocols for stable production in CHO cells are described in this unit. Typical methods for transfection using commercially available plasmid expression vectors are described, along with methods to select for stable expression and methods for amplifying the expression level in the transfected cell. Following this, procedures are presented for efficient cell growth to obtain significant amounts of protein product. Support protocols describe freezing of cells, determination of growth rates, determination of specific productivity of cells, preparing samples for assay, and setting up a 10‐day shaker‐flask growth curve.
Characterizing Recombinant Proteins Using HPLC Gel Filtration and Mass Spectrometry
Authors: Begg Gillian E. , Harper Sandra L. , Speicher David W. 
Published: June 2004

AbstractRecombinant proteins are subject to many forms of heterogeneity, including aggregation, proteolytic degradation, chemical modification, mutation, and incorrect translation. This unit describes methods for the detection and identification of these problems using analytical HPLC gel filtration and MALDI‐MS. Preliminary characterization of recombinants is necessary before the structure or function of the protein can be investigated.
Hydroxylapatite Chromatography
Authors: Broadhurst Anne V. 
Published: June 2004

AbstractHydroxylapatite (also called hydroxyapatite), a form of calcium phosphate, can be used as a matrix for the chromatography of both proteins and nucleic acids. Protocols are provided for both standard low‐pressure chromatography of a protein mixture using a hydroxylapatite column prepared in the laboratory, and an HPLC method, applicable to proteins and nucleic acids, that uses a commercially available column. Alternate protocols describe column chromatography using a step gradient or batch binding and step‐gradient elution.
Lectin Affinity Chromatography
Authors: Freeze Hudson H. 
Published: June 2004

AbstractThis unit describes the use of lectins for preparative glycoprotein purification. Con A‐Sepharose and WGA‐agarose are used for convenience and availability. Instructions are given for a small‐scale pilot procedure to test for lectin binding and to determine elution conditions. There are many variations on the basic procedure in the literature, but all use the same principles: bind the protein to immobilized lectin through its sugar chain, wash away unbound protein, and elute bound protein with a simple sugar that resembles the sugar ligand of the bound protein.
Metal‐Chelate Affinity Chromatography
Authors: Petty Kevin J. 
Published: June 2004

AbstractRecombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxyl terminus can be purified using a resin containing nickel ions (Ni2+) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique, known as metal‐chelate affinity chromatography (MCAC), can readily be performed with either native or denatured protein. This unit discusses techniques for creating a fusion protein consisting of the protein of interest with a histidine tail attached. A procedure for xpression of histidine‐tail fusion proteins and their purification in native form by MCAC is described, and two alternate protocols describe purification of histidine‐tail fusion proteins by MCAC under denaturing conditions and their renaturation by either dialysis or solid‐phase renaturation. Support protocols are provided for analysis of the purified product and regeneration of the NTA resin. All of these protocols are easily adaptable to any protein expression system.
Immunoaffinity Chromatography
Authors: Springer Timothy A. 
Published: June 2004

AbstractThis unit describes the isolation of soluble or membrane‐bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. This technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. Specifically, antibodies are coupled to Sepharose (an insoluble, large‐pore‐size chromatographic matrix). High‐molecular‐weight antigens pass freely into and out of the pores and bind to antibodies covalently bound to the matrix. To elute the bound antigen from the immunoaffinity matrix, the antibody‐antigen interaction is destabilized by brief exposure to high‐ or low‐pH buffer. Batch purification of antigens is provided as an alternate procedure that shortens the column loading time. The detergent octyl β‐D‐glucoside can be used instead of Triton X‐100 for elution. Because octyl β‐D‐glucoside has a high critical micelle concentration (CMC), a protocol is provided for its removal by dialysis. The procedure for covalently linking an antibody to Sepharose using the cyanogen bromide activation method is given in a support protocol.
Removal of N‐Terminal Blocking Groups from Proteins
Authors: Fowler Elizabeth , Moyer Mary , Krishna Radha G. , Chin Christopher C.Q. , Wold Finn 
Published: June 2004

AbstractTwo enzymatic methods commonly used in N‐terminal sequence analysis of blocked proteins are presented in this unit; one uses pyroglutamate aminopeptidase for Nα‐pyrrolidone carboxyl‐proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl‐peptide hydrolase for Nα‐acyl‐proteins blocked with other acyl groups. A describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl‐peptide hydrolase must include fragmentation of the protein before unblocking can be carried out, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N‐terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Protocols are also provided for unblocking N‐terminally blocked proteins using acid‐catalyzed hydrolysis or methanolysis, hydrazinolysis, and β‐elimination after acid‐catalyzed N‐O shift. Alternate protocols describe chemical removal of acetyl and longer‐chain alkanoyl groups, as well as formyl groups to open the cyclic imide of pyrrolidone carboxylate.
C‐Terminal Sequence Analysis
Authors: Bergman Tomas , Cederlund Ella , Jörnvall Hans , Fowler Elizabeth 
Published: June 2004

AbstractCarboxy‐terminal (C‐terminal) sequence analysis is used for direct confirmation of the C‐terminal sequence of native and expressed proteins, for detection and characterization of protein processing at the C‐terminus, for identification of post‐translational proteolytic cleavages, and for obtaining partial sequence information on N‐terminally blocked protein samples in order to facilitate design of oligonucleotide probes for gene cloning. This unit describes an automated chemical method and a manual enzymatic (carboxypeptidase digestion) method for determining C‐terminal sequence information. Carboxypeptidase digestion requires only a standard amino acid analysis method.
Detection of Phosphorylation by Enzymatic Techniques
Authors: Shenolikar Shirish 
Published: June 2004

AbstractThis unit presents protocols for examining the functional effects elicited by nonspecific acid or alkaline phosphatases that dephosphorylate many phosphoproteins in vitro. 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.
Analysis of Disulfide Bond Formation
Authors: Braakman Ineke , Hebert Daniel N. 
Published: June 2004

AbstractIn this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein is then chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS‐PAGE with and without prior reduction. The difference in mobility observed between the gels with unreduced and reduced samples is due to disulfide bonds in the unreduced protein.
Analysis of Protein Acylation
Authors: Jackson Caroline S. , Magee Anthony I. 
Published: June 2004

AbstractProtein acylation is the covalent attachment of fatty acids to a protein; the most commonly added fatty acids are myristate (14:0) and palmitate (16:0). In this unit, protocols describe the use of radiolabeled fatty acids to label eukaryotic cells in vitro. The radiolabeled material produced can then be analyzed by the various methods described here: determination of the type of fatty acid linkage, checking for interconversion by determining the nature of the protein‐bound label, and identification of the protein‐bound fatty acid.
Analysis of Oxidative Modification of Proteins
Authors: Yan Liang‐Jun , Sohal Rajindar S. 
Published: June 2004

AbstractProtein oxidation has been implicated in a variety of degenerative diseases as well as in the aging process. This unit describes techniques for the quantification of various protein oxidation products, including protein carbonyls, loss of protein thiol groups, dityrosine and nitrotyrosine, and isoaspartate formation. Such oxidatively modified products may also be used as biomarkers for the assessment of oxidative stress during aging and/or disease.
Searching Sequence Databases Over the Internet: Protein Identification Using MS‐Tag
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L. 
Published: June 2004

AbstractAlthough peptide mass mapping is fast and simple, its success can be compromised by the purity of the protein in one gel spot, errors in the sequence database, and the detection of too few peptides in the MS map of a given protein to permit reliable database matching. In those cases, partial amino acid sequence that is determined using post‐source decay (PSD) analysis or tandem mass spectrometry is required to establish the correct protein identification among possibilities suggested by MS‐Fit. Post‐source decay (PSD) involves the detection of the fragmentation product ions (metastable ions) of a selected mass value window containing the precursor ion chosen, that occur in the field‐free region between the ion source and the reflectron. This unit covers some of the practical issues in obtaining and analyzing a typical PSD spectrum using MS‐Tag, a database interrogation program that enables the identification of proteins with a limited number of peptide fragment ions by matching existing database sequences. It is a user‐friendly program that simultaneously considers a variety of ion types, but still allows single‐mismatch‐tolerant searching. Although MALDI‐PSD database searching is a widely used tool, an alternative method also described in this unit is analysis by MALDI collision‐induced dissociation (MALDI‐CID).
Overview of Protein Structural and Functional Folds
Authors: Sun Peter D. , Foster Christine E. , Boyington Jeffrey C. 
Published: June 2004

AbstractThis overview provides an illustrated, comprehensive survey of some commonly observed protein‐fold families and structural motifs, chosen for their functional significance. It opens with descriptions and definitions of the various elements of protein structure and associated terminology. Following is an introduction into web‐based structural bioinformatics that includes surveys of interactive web servers for protein fold or domain annotation, protein‐structure databases, protein‐structure‐classification databases, structural alignments of proteins, and molecular graphics programs available for personal computers. The rest of the overview describes selected families of protein folds in terms of their secondary, tertiary, and quaternary structural arrangements, including ribbon‐diagram examples, tables of representative structures with references, and brief explanations pointing out their respective biological and functional significance.
Disulfide Bond Formation in Peptides
Authors: Chen Lin , Annis Ioana , Barany George 
Published: June 2004

AbstractThe formation of disulfide bridges is often a crucial final stage in peptide synthesis. There is compelling evidence that the disulfide pattern can be critical in the folding and structural stabilization of many natural peptide and protein sequences, while the artificial introduction of disulfide bridges into natural or designed peptides may often improve biological activities/specificities and stabilities. This unit provides a highly selective, albeit state‐of‐the‐art, menu of procedures that can be performed to establish intramolecular or intermolecular disulfide bridges in targets of varying complexities.
Overview of the Quantitation of Protein Interactions
Authors: Philo John S. 
Published: June 2004

AbstractThe biological function of many proteins involves reversible interactions with other proteins, nucleic acids, or other non‐protein ligands. Such interactions play many different roles in a wide range of cellular processes. A few examples are: (1) storing or transporting key metabolites (e.g., O2 storage by myoglobin); (2) forming and maintaining the quaternary structure of multi‐subunit enzymes; (3) specific binding and recognition events (antigen‐antibody, hormone‐receptor, transcription factor‐promoter); and (4) self‐assembly of large structures (microtubules, chromatin). Thus, the quantitative characterization of such interactions represents an important part of understanding the function of such proteins and their role in these cellular events. This unit sets the tone for the rest of the chapter, and gives important information necessary to understand some of the topics that will be covered in future supplements, such as sedimentation equilibrium (analytical and micro‐preparative), surface plasmon resonance (SPR), size‐exclusion chromatography (SEC) with on‐line light scattering, and chemical cross‐linking.