bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis of Multiple Peptides on Plastic Pins
Authors: Rodda Stuart J. 
Published: June 2004

AbstractThis unit covers the strategy of the multiple peptide approach to biological scanning, the synthetic protocols, and the handling of peptides after synthesis: cleavage, preliminary purification, storage, and analysis. It is specific for the multipin technique using equipment obtained from Chiron Technologies, although some of the approaches are applicable to other multiple synthesis techniques. This unit also includes protocols for preparing Fmoc‐amino acid solutions and for acetylating or biotinylating synthesized peptides.
Synthesis and Application of Peptide Dendrimers As Protein Mimetics
Authors: Tam James P. , Spetzler Jane C. 
Published: June 2004

AbstractThe use of peptides to mimic a portion of a protein structure is a challenging and powerful tool in the discovery of new drugs. In native proteins, discontinuous bioactive peptide surfaces are held together in a particular conformation by the structural rigidity of the protein. Approaches to mimicking a structural surface center on bringing the potential peptide sequences together by assembling the peptide chains on a template. These templates can be flexible dendrimeric or cyclic peptides as well as more rigid organic molecules. The Multiple Antigen Peptide (MAP) system represents a novel approach to preparing peptide immunogens. The MAP consists of an inner core matrix built up of a large layer of Lys residues and a surface of peptide chains attached to the core matrix. Because of its dendrimeric structure, MAP can be very useful as a template for assembling potential peptide surfaces. A variation of this procedure, the cyclic Multiple Antigen Peptide (cMAP) approach, is also presented here. Having branched multiple closed‐chain architectures, the cMAP system is often a superior approach for protein mimetics because the multiple constrained peptides can mimic bioactive conformations. Whether to select this approach over MAP depends on the properties of the peptides, but usually if the peptides are too small to adopt a stable conformation on their own, incorporation of a cyclic structure may be necessary. MAPs have been applied to areas of study such as inhibitors, artificial proteins, affinity purifications, and intracellular transport.
Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins
Authors: Golemis Erica A. , Serebriiskii Ilya , Finley Russell L. , Kolonin 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.
Phage‐Based Expression Cloning to Identify Interacting Proteins
Authors: Stone Julie M. 
Published: June 2004

AbstractInteraction cloning (also known as expression cloning) is a technique to identify and clone genes which encode proteins that interact with a protein of interest, or “bait” protein. The procedure presented in this unit involves a fusion protein consisting of bait protein and glutathione‐S‐transferase (GST) with a protein kinase A site at the junction between them (the protocol can, however, be adapted to use other PKA‐containing recombinant proteins). The labeled protein is subsequently used as a probe to screen a l bacteriophage‐derived cDNA expression library, which expresses
b ‐galactosidase fusion proteins that contain in‐frame gene fusions. The phages lyse cells, form plaques, and release fusion proteins that are adsorbed onto nitrocellulose membrane filters. The filters are blocked with excess nonspecific protein to eliminate nonspecific binding and probed with the radiolabeled bait protein. This procedure leads directly to the isolation of genes encoding the interacting protein, bypassing the need for purification and microsequencing or for antibody production.
High‐Throughput Screening for Protein‐Protein Interactions Using Yeast Two‐Hybrid Arrays
Authors: Cagney Gerard , Uetz Peter 
Published: June 2004

AbstractArrays are used for parallel assays of large numbers of proteins in a single experiment. They provide an alternative to libraries for protein screening purposes. This unit describes a protein array composed of living yeast (Saccharomyces cerevisiae) cells that can be used in a functional screen for protein interactions, the two‐hybrid assay. The protocols described here use the yeast proteome as an example, but they can be applied to any other genome or subset thereof.
Reduced‐Scale Large‐Zone Analytical Gel‐Filtration Chromatography for Measurement of Protein Association Equilibria
Authors: Beckett Dorothy 
Published: June 2004

AbstractThe proteasome plays a central role in eukaryotic cells since it is responsible for the degradation of specific proteins involved in a large range of cellular processes. Analysis of proteasome mechanisms of action, or in vitro reconstitution, or dissection of the complex biological pathways in which it partakes, requires a reliable source of pure active proteasome. Although the biologically relevant form of the proteasome is usually considered to be the 26S proteasome, this unit describes different methods for purification and study of both 26S and 20S proteasomes from
Saccharomyces cerevisiae cells.
Proteases
Authors: Barrett Alan J. 
Published: June 2004

AbstractThe processes of growth and remodeling of cells and tissues in multicellular organisms require the breakdown of old protein molecules, in concert with the synthesis of new ones. For example, many newly‐synthesized molecules require proteolytic processing to convert them to biologically active forms. Proteolysis can terminate the activity of a protein—e.g., capsases mediate apoptosis, which is a vital step in the life cycle of the cell. Proteolysis contributes to defense systems too, as the recognition of peptide fragments of foreign proteins triggers the immune response. Proteases are the class of enzymes involved in these important reactions. This unit discusses the general categories of proteases, and sets the stage for addition of overview units on cysteine proteases, aspartic proteases, and metalloproteases, as well as protocol units featuring techniques for analyzing mammalian and yeast proteasomes and protease inhibitors, among other topics.
Papain‐like Cysteine Proteases
Authors: Brömme Dieter 
Published: June 2004

AbstractThe name “cysteine protease“ refers to the protease's nucleophilic cysteine residue that forms a covalent bond with the carbonyl group of the scissile peptide bond in substrates. The papain‐like cysteine proteases, classified as the “C1 family” are the most predominant cysteine proteases. These proteases are found in viruses, plants, primitive parasites, invertebrates, and vertebrates alike. Mammalian papain‐like cysteine proteases are also known as cathepsins. This unit discusses cathepsins, and their subcellular and tissue localization, catalytic mechanism, and substrate specificity. Several tables illustrate the properties of the various cathepsins.
Metalloproteases
Authors: Nagase Hideaki 
Published: June 2004

AbstractMetalloproteases (metallopeptidases) are composed of a diverse group of endopeptidases and exopeptidases. Because metalloproteases play key roles in many normal biological processes, their aberrant activities have been implicated in diseases such as arthritis, cancer, cardiovascular diseases, nephritis, disorders in the central nervous system, fibrosis, and infection, as well as others. This unit provides an overview of metalloproteases, including their classification, catalytic mechanisms, structures, substrate specificities, and common inhibitors.
Purification and Characterization of Proteasomes from Saccharomyces cerevisiae
Authors: Glickman Michael , Coux Olivier 
Published: June 2004

AbstractThe proteasome plays a central role in eukaryotic cells since it is responsible for the degradation of specific proteins involved in a large range of cellular processes. Analysis of proteasome mechanisms of action, or in vitro reconstitution, or dissection of the complex biological pathways in which it partakes, requires a reliable source of pure active proteasome. Although the biologically relevant form of the proteasome is usually considered to be the 26S proteasome, this unit describes different methods for purification and study of both 26S and 20S proteasomes from Saccharomyces cerevisiae cells.
Purification of the Eukaryotic 20S Proteasome
Authors: Wilk Sherwin , Chen Wei‐Er 
Published: June 2004

AbstractThe 20S proteasome is the catalytic core of the major extralysosomal proteolytic system of the cell. Combination of the 20S proteasome with a complex of regulatory proteins forms the 26S proteasome, which in turn is responsible for the recognition and degradation of ubiquitin‐protein conjugates. As described in this unit, the constitutive form of the 20S proteasome can be conveniently purified as a stable and homogeneous preparation from bovine pituitaries. A support protocol details an enzyme assay used in evaluating proteasomal activity. The 20S proteasome is the catalytic core of the major extralysosomal proteolytic system of the cell.
Expression, Purification, and Characterization of Aspartic Endopeptidases:PlasmodiumPlasmepsins and “Short” Recombinant Human Pseudocathepsin
Authors: Beyer Bret B. , Goldfarb Nathan E. , Dunn Ben M. 
Published: June 2004

AbstractThe unit describes a basic protocols utilized to obtain milligram amounts of enzymatically active, pure recombinantPlasmodiumplasmepsins and "short" human pseudocathepsin D. Specific details for the expression and purification ofPlasmodium falciparumplasmepsin 2 and "short" human pseudocathepsin D in zymogen form are described in this chapter. The plasmepsin 2 protocols are also applicable toPlasmodium vivax, P. ovale, andP. malariaeplasmepsins, as well asP. falciparumplasmepsin 4.
Isolation of Organelles and Prefractionation of Protein Extracts Using Free‐Flow Electrophoresis
Authors: Weber Peter J. A. , Weber Gerhard , Eckerskorn Christoph 
Published: June 2004

AbstractOne of the major obstacles in the analysis of proteomes is the extreme complexity of any particular cell or biological fluid. Free‐flow electrophoresis (FFE) is a powerful tool for reduction of this complexity, which is a prerequisite for systematic and comprehensive protein analyses. Protocols are provided in this unit for sample fractionation at two different stages: on the protein level by isoelectric focusing FFE fractionation of crude protein mixtures such as whole cell lysates, and on a subcellular level by zone‐electrophoretic FFE purification of organelles.
Quantitative Protein Profile Comparisons Using the Isotope‐Coded Affinity Tag Method
Authors: Yi Eugene C. , Goodlett David R. 
Published: June 2004

AbstractCurrent methods for measuring pairwise changes in protein expression involve differential stable isotopic labeling of proteins or peptides either in vivo or in vitro followed by identification and quantification using a mass spectrometer. In these methods, the mass spectrometer detects two different masses, which correspond to a single protein from two different samples that have been labeled with either a heavy or normal isotope. Changes in protein expression are observed when the identical peptide from each of two biological conditions is identified and a difference is detected in the measurements comparing the peptide labeled with the heavy isotope to the one with a normal isotopic distribution. This approach allows the simultaneous comparison of the expression of many proteins between two different biological states (e.g., yeast grown on galactose versus glucose, or normal versus cancer cells). This unit describes one of these popular methods for quantitative protein profiling using the isotope‐coded affinity tag (ICAT) technique.
Laboratory Safety
Authors: Lunn George , Lawler (chemical resistance of gloves) Gretchen 
Published: June 2004

AbstractThis appendix provides protocols for some commonly used disposal and decontamination procedures along with analytical techniques that are used to verify that reagents have been decontaminated. Some of the specific reagents covered are diaminobenzidine, ethidium bromide, cyanogen bromide and chloromethylsilane. With modification, these assays may also be used to determine the concentration of a particular chemical. Precautions are also detailed for routine handling of viable pathogenic microorganisms, as well as all human‐derived materials, because they may harbor dangerous pathogens such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), cytomegalovirus (CMV), Epstein‐Barr virus (EBV), and a host of bacterial pathogens.
Statistics: Detecting Differences Among Groups
Authors: Robakiewicz Phil , Ryder Elizabeth F. 
Published: June 2004

AbstractSome of the statistical tests most commonly used (and misused) in biological research are presented here. The tests discussed are those used for comparisons among groups (e.g., t test and ANOVA). The purpose of this appendix is to enable the investigator to determine rapidly the most appropriate way to analyze data, and to point out some of the most common errors to avoid.
Analyzing Radioligand Binding Data
Authors: Motulsky Harvey , Neubig Richard 
Published: June 2004

AbstractA radioligand is a radioactively labeled drug that can associate with a receptor, transporter, enzyme, or any protein of interest. Measuring the rate and extent of binding provides information on the number of binding sites, and their affinity and accessibility for various drugs. Radioligand binding experiments are easy to perform, and provide useful data in many fields. For example, radioligand binding studies are used to study receptor regulation, investigate receptor localization in different organs or regions using autoradiography, categorize receptor subtypes, and probe mechanisms of receptor signaling. This unit reviews the theory of receptor binding and explains how to analyze experimental data. Since binding data are usually best analyzed using nonlinear regression, this unit also explains the principles of curve fitting with nonlinear regression.
Southern Blotting
Authors: Brown Terry 
Published: June 2004

AbstractSouthern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high‐salt transfer buffer to promote binding of DNA to the membrane. With the high‐salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. Immobilization is achieved by UV irradiation (for nylon) or baking (for nitrocellulose). A describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. The advantage of this combination is that no post‐transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. The method can also be used with neutral nylon membranes but less DNA will be retained. A second alternate protocol describes a transfer method based on a different transfer‐stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane described in the first basic and alternate protocols has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid than the basic protocol and can result in more complete transfer. Although the ease and reliability of capillary transfer methods makes this far and away the most popular system for Southern blotting with agarose gels, it unfortunately does not work with polyacrylamide gels, whose smaller pore size impedes the transverse movement of the DNA molecules. The third alternate protocol describes an electroblotting procedure that is currently the most reliable method for transfer of DNA from a polyacrylamide gel. Dot and slot blotting are also described.
Hybridization Analysis of DNA Blots
Authors: Brown Terry , Struhl Kevin 
Published: June 2004

AbstractRestriction endonucleases recognize short DNA sequences and cleave double‐stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. This appendix describes restriction endonucleases and their properties.
Receptor Theory
Authors: Kenakin Terry 
Published: June 2004

AbstractThe equations and models that describe the interaction of chemicals with receptors comprise what is generally known as drug‐receptor theory. Although largely adapted from enzyme kinetics, the principles necessarily differ from those of enzyme‐substrate interactions in that they deal with chemicals that are not changed by the receptor, but rather interact in a reversible manner to produce a change in the state of the receptor that is then transmitted to the cellular host. This overview describes the evolution of drug receptor theory, including occupancy theory, two‐state theory, and the ternary complex model.
Characterization of Neuropeptide Y (NPY) Receptors
Authors: Karbon William , Marasco Julie , Hale Clarence 
Published: June 2004

AbstractNeuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) constitute a trio of structurally related peptides that elicit a variety of physiological responses in targets including the brain, gut, and vascular smooth muscle. These actions are mediated by a family of G protein‐coupled receptors, each of which exhibits a distinct pharmacological fingerprint. This unit describes protocols for identifying and characterizing the pharmacological properties of two NPY receptor subtypes, Y1 and Y2. At least four other members of this receptor family have been described (Y3, Y4, Y5 and y6), and a brief description of these receptors and relevant literature citations are also provided.
Radioligand Binding Assays for the Glycine Site on N‐Methyl‐D‐Aspartate Receptors
Authors: Perschke Scott , Ferkany John 
Published: June 2004

AbstractThis unit describes a competitive binding assay for the glycine binding site on the NMDA subtype of glutamate receptors in rat brain homogenates. Agonists of the NMDA receptor associated glycine binding site have been proposed as potential therapeutics in cognitive disorders. Conversely, antagonists may be useful in a variety of disorders associated with excessive activation of EAA receptors, including Parkinsons, Huntingtons and Alzheimers Diseases, and neuropathic pain, among others. This unit describes a competitive binding assay for the glycine binding site on the NMDA subtype of glutamate receptors in rat
Characterization of Neurotensin Receptors
Authors: Kitabgi Patrick , Pélaprat Didier 
Published: June 2004

AbstractThis unit describes procedures for performing competition binding assays with neurotensin receptor subtypes 1 and 2 (NTS1 and NTS2). Binding assays using cloned receptors, brain membranes, and primary cultured mesencephalic neurons are presented. NTS1 binding assays employing either radiolabeled neurotensin or SR 48692 (a nonpeptide neurotensin antagonist) as radioligands are described. These procedures may be used to screen selective ligands at neurotensin receptor subtypes.
Agonist‐Stimulated [35S]GTPγS Binding
Authors: Alper Richard H. 
Published: June 2004

AbstractThis unit presents a convenient method for studying receptor function by using a binding assay to assess the initial step in receptor‐mediated G protein activation in membranes prepared from rat brain. While the example procedure presented in this unit employs receptor agonists for serotonin1A receptors in the hippocampus, it can be employed to study the activity of any substance that activates a receptor coupled to a G protein, particularly GI. Alternate protocols describe modifications that can be used to simplify and increase the accuracy of the basic protocol, as well as how to use the basic protocol to study receptor antagonists.
In Vitro Enzymatic Assays for Ser/Thr‐Selective Protein Kinases
Authors: Bramson H. Neal 
Published: June 2004

AbstractMedium and high throughput methods for measuring the activities of Ser/Thr‐selective protein kinases are described. These methods utilize radiochemical detection, fluorescence polarization, and ultraviolet spectroscopy to monitor transfer of the gamma‐phosphoryl group of ATP to protein or peptide substrates. These assays have utility in characterizing protein kinase inhibitors and in mechanistic studies.
Overview of Length‐Tension Relationships in Isolated Tissue
Authors: Kenakin Terry 
Published: June 2004

AbstractThis unit describes critical aspects of preparing isolated tissues for experiments to determine the response of specific receptors in the tissue to compounds in a surrounding fluid bath. The two most common methods of evaluating muscle function in pharmacological length‐tension relationships experiments is through measuring isometric and isotonic contraction. In an isometric contraction, which occurs in muscles when they exert force without changing length, the contractile element shortens and the stress transmitted onto the series elastic component is measured in newtons or grams, reflecting the increase in total tension. Isotonic contraction is the shortening of a muscle under a constant load. A key factor in the sensitivity of an isolated tissue receptor system is the resting length under which the muscle is mounted. A discussion of important issues relating to resting length is included.
Neuropeptide Y (NPY) Receptor Assays
Authors: Daniels Alex , Harrington Wallace 
Published: June 2004

AbstractNeuropeptide Y (NPY) is a very abundant neuropeptide that has been implicated in the regulation of a wide variety of physiological functions such as vascular tone, food ingestion, mood, and hormone secretion. In the kidney, NPY is a potent vasoconstrictor, localized predominantly in adrenergic nerve terminals supplying the afferent arteriole and the juxtaglomerular apparatus. Therefore, isolated rat kidney provides a convenient model for examining the vasoconstriction produced by NPY and its analogs. This unit describes a general method for the preparation, handling, and perfusion of the isolated rat kidney, along with measurement of the tissue response upon exposure to NPY (or its analogs) and NPY antagonists. Neuropeptide Y (NPY) is a very abundant neuropeptide that has been implicated in the regulation of a wide variety of physiolog.