bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic Digestion of Proteins on PVDF Membranes
Authors: Fernandez Joseph , Mische Sheenah M.
Published: June 2004

Enzymatic digestion of membrane‐bound proteins is one of the most widely used procedures for determining the internal amino acid sequence of proteins that either have a blocked amino terminus or require two or more stretches of sequence data for DNA cloning or confirmation of protein identification. Because the final step of protein purification is usually SDS‐PAGE, electroblotting to either polyvinylidene difluoride (PVDF) or nitrocellulose is the simplest and most common procedure for recovering protein free of contaminants (e.g., SDS or acrylamide) with a high yield. As described in this unit, PVDF is preferred over nitrocellulose because it can be used for a variety of other structural analysis procedures, such as amino‐terminal sequence analysis and amino acid analysis. In addition, peptide recovery from PVDF membranes is higher than from nitrocellulose, particularly from higher‐retention PVDF (e.g., ProBlott, Transblot, Westran, or Immobilon Psp). Finally, PVDF‐bound protein can be stored dry, as opposed to nitrocellulose‐bound protein, which must remain wet during handling and storage to prevent loss of peptides during digestion.
Reversed‐Phase Isolation of Peptides
Authors: Henzel William J. , Stults John T.
Published: June 2004

Reversed‐phase high‐performance liquid chromatography (HPLC) is a fundamental tool for the isolation and analysis of peptides. Peptides are separated on a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent concentration. This unit presents protocol for separation of 5‐ to 500‐pmol of peptides on a narrow‐bore (2‐mm‐i.d.) or microbore (1‐mm‐i.d.) column. Smaller quantities can be separated capillary HPLC columns, as described. Capillary HPLC columns, however, require a gradient flow rate of 3 to 5 ml/min, which most current HPLC pumps cannot attain without modifications. A procedure is therefore provided for constructing a capillary HPLC system using readily available components. HPLC peaks that appear to be symmetrical may actually contain coeluting peptides. Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry and capillary electrophoresis are described for analysis of a small portion of an HPLC fraction to determine the number of components present in a small sample. These methods can be utilized to screen fractions prior to automated sequencing.
N‐Terminal Sequence Analysis of Proteins and Peptides
Authors: Reim David F. , Speicher David W.
Published: June 2004

Amino‐terminal (N‐terminal) sequence analysis is used to identify the order of amino acids of proteins or peptides, starting at their N‐terminal end. This unit describes the sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Perkin‐Elmer Procise Sequencer. Sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Hewlett‐Packard Model G1005A sequencer is also described. Methods are provided for optimizing separation of PTH amino acid derivatives on Perkin‐Elmer instruments and for increasing the proportion of sample injected onto the PTH analyzer on older Perkin‐Elmer instruments by installing a modified sample loop. The amount of data obtained from a single sequencer run is substantial, and careful interpretation of this data by an experienced scientist familiar with the current operation performance of the instrument used for this analysis is critically important. A discussion of data interpretation is therefore provided. Finally, discussion of optimization of sequencer performance as well as possible solutions to frequently encountered problems is included.
Inhibition of N‐Linked Glycosylation
Authors: Powell Leland D.
Published: June 2004

This unit describes the use of inhibitors in cultured cells to prevent N‐linked glycosylation of proteins to yield glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of oligosaccharides on specific proteins or intact 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 [3H]mannose using TCA precipitation or endo H digestion. A support protocol details a method for concentrating proteins by acetone precipitation.
Detection of Glycophospholipid Anchors on Proteins
Authors: Doering Tamara L. , Englund Paul T. , Hart Gerald W.
Published: June 2004

Many 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 partitioning of total cellular and isolated proteins with Triton X‐114 is described in this unit, and precondensation of Triton X‐114, which is necessary to remove hydrophilic contaminants before partitioning, is outlined in a. The protein may also be subjected to specific enzymatic or chemical cleavages to release it from its GPI anchor. Phospholipase cleavage (starting with intact cells or membranes, or with isolated protein) is detailed, and chemical cleavage with nitrous acid is also described. If GPI‐anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. A protocol for separation of lipid moieties released from proteins is provided and base hydrolysis of proteins is also presented.
Detection and Analysis of Proteins Modified by O‐Linked N‐Acetylglucosamine
Authors: Zachara Natasha E. , Cole Robert N. , Hart Gerald W. , Gao Yuan
Published: June 2004

First, a protocol for increasing the stoichiometry of O‐GlcNAc on proteins is given. This is followed by simple techniques for the detection/screening of O‐GlcNAc‐modified proteins either by immunoblotting or lectin affinity chromatography. Separate protocols verify that the glycan is O‐linked GlcNAc. These methods are followed by protocols for more comprehensive analysis of O‐GlcNAc modified proteins, including labeling of O‐GlcNAc residues with [3H]Gal, and subsequent product analysis. The final two protocols assay for O‐GlcNAc transferase and O‐GlcNAcase activity, respectively.
Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
Authors: Sefton Bartholomew M.
Published: June 2004

This unit describes 32PI labeling and lysis of cultured cells to be used for subsequent immunoprecipitation of proteins. The procedure is suitable for insect, avian, and mammalian cells and can be used with both adherent and nonadherent cultures. The protocol 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.
Phosphopeptide Mapping and Identification of Phosphorylation Sites
Authors: Meisenhelder Jill , Hunter Tony , van der Geer Peter
Published: June 2004

Protein phosphorylation is a common modification for many proteins in the cell. Phosphorylation can affect localization of a protein, its stability, and its ability to dimerize or form stable complexes with other molecules. To understand the underlying mechanisms behind the phosphorylation of a given protein, it is often necessary to precisely identify which amino acid residues are phosphorylated. This unit describes the technique of phosphopeptide mapping. In this procedure, a radiolabeled protein is proteolytically digested, and the resulting phosphopeptides are separated in two dimensions on a TLC plate. The phosphopeptides are also analyzed by HPLC and mass spectrometry or peptide microsequencing. Such mapping gives information about the number of phosphorylation sites present in the protein, and can also be used to find out if sites of phosphorylation on a protein change upon treatment of cells with specific agents.
Analysis of Protein Ubiquitination
Authors: Laney Jeffrey D. , Hochstrasser Mark
Published: June 2004

Attachment of ubiquitin (Ub) to a protein requires a complex of enzymes that recognize the substrate and promote Ub transfer. Sequence motifs present in these enzymes may indicate that other uncharacterized proteins containing these motifs have a biochemical function of Ub‐protein ligation, and several in vitro methods are described in this unit for determining if a protein has Ub‐transferring activity. They include psmunoblotting of psmunoprecipitated proteins, affinity purification using His‐tagged ubiquitin, assaying for auto‐ubiquitination of E3, and assaying ubiquitination of a model substrate protein. These methods are suitable for a variety of eukaryotic cells, but techniques are specifically described for use with yeast and mammalian cells.
Modification of Cysteine
Authors: Crankshaw Mark W. , Grant Gregory A.
Published: June 2004

This unit describes a number of methods for modifying cysteine residues of proteins and peptides by reduction and alkylation procedures. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N‐ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and
N‐(iodoethyl)‐trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4‐vinylpyridine and acrylamide, are described, and a specialized procedure for 4‐vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is detailed. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples.
Modification of Amino Groups
Authors: Geoghegan Kieran F.
Published: June 2004

This unit describes group‐specific modifications of amino groups. These reactions remain valid tools for early‐stage evaluation of structure‐function relationships, but are now valued even more for their applications in the preparation of bioconjugates, affinity columns, biosensors, and tagged macromolecules. Protocols are provided here for reaction of amino groups with succinimidyl esters and isothiocyanates. These methods are broadly useful for the stable coupling to proteins of groups with useful, non‐native functional properties. These include biotin for detection or recovery, fluorescent groups for biophysics or cytochemistry, cross‐linking reagents for making bioconjugates, or metal‐chelators that permit proteins to be loaded with radioisotopes for medical imaging or antitumor therapy. These applications require accurate product characterization, which preferably is performed by mass spectrometry, as described in this unit as a support procedure. A protocol employing succinic or acetic anhydrides to change the charge state of protein amino groups is provided here, as is a procedure for reductive alkylation that leaves their charge unaltered but converts primary amines to secondary or tertiary amines.
Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Analysis of Peptides
Authors: Henzel William J. , Stults John T.
Published: June 2004

Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) is one of the most useful techniques for determining the mass of biomolecules, with exceptional capabilities for mass analysis of peptides. Relative to other ionization techniques, it provides high sensitivity and excellent tolerance of salt and other common buffer components. Routine detection limits for peptides are in the subpicomole range. The ions commonly observed are the protonated molecules (M+H+), which makes data analysis relatively easy. This overview discusses instrument configuration and calibration, sample preparation, along with specific approaches for analyzing peptide mixtures, synthetic peptides, and chemical modifications of peptides.
In‐Gel Digestion of Proteins for MALDI‐MS Fingerprint Mapping
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L.
Published: June 2004

Mass spectrometry (MS) has emerged as a sensitive, versatile, and rapid method for protein identification, following the advent of electrospray ionization mass spectrometry (ESI‐MS) and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). The advantages of MALDI‐MS over ESI‐MS include its relatively high tolerance to contamination from biological matrices, its high sensitivity, the relative ease of interpreting spectra from mixtures, and the formation of singly protonated molecular ions for tandem analysis. Peptide fingerprint mass mapping and partial peptide sequencing using post‐source decay and ladder sequencing by MALDI‐MS in combination with algorithms for sequence database interrogation have the potential for identification and structural investigation of proteins. This unit describes in‐gel digestion for peptide mass mapping of picomole to subpicomole quantities of protein derived from Coomassie‐ or silver‐stained polyacrylamide gels. After digestion, the peptides are extracted from the gel and mass analyzed.
De Novo Peptide Sequencing via Manual Interpretation of MS/MS Spectra
Authors: Addona Terri , Clauser Karl
Published: June 2004

Databases of complete genome sequences are rapidly appearing, and a recent trend in interpretation of peptide MS/MS spectra is database matching. A caveat of database matching is that it can only be successful if the database being searched contains the identical amino acid sequence, or one with high homology, to the peptide being investigated. In instances where high‐quality MS/MS spectra are obtained for which there is no corresponding database match (e.g., when the peptide of interest is derived from a novel protein from an organism whose genome is not known, has post‐translationally modified amino acids, or has multiple amino acid substitutions as compared to the homologous sequence from a different species), de novo (or manual) interpretation of the MS/MS spectrum, as described here, is required. Methods are also provided for verification of the results of the de novo analysis through chemical modification and limited manual Edman degradation of the peptide.
Overview of Macromolecular Electron Microscopy: An Essential Tool in Protein Structural Analysis
Authors: Steven Alasdair C.
Published: June 2004

This unit aims to overview the application of NMR spectroscopy to proteins. It is not intended to provide an exhaustive “how to” guide, but rather to give a flavor of both well‐established and emerging NMR techniques used in the elucidation of protein structure. It starts with a brief introduction to the basic principles of NMR and the information provided by this technique, and goes on to discuss the instrumentation involved, spectral assignment methods for small and large proteins, and the utility of other spin active nuclei (e.g., 13C and 15N) to aid assignment of the latter.
Crystallization of Macromolecules
Authors: Messick Troy , Marmorstein Ronen
Published: June 2004

X‐ray crystallography has evolved into a very powerful tool to determine the three‐dimensional structure of macromolecules and macromolecular complexes. The major bottleneck in structure determination by X‐ray crystallography is the preparation of suitable crystalline samples. This unit outlines steps for the crystallization of a macromolecule, starting with a purified, homogeneous sample. The first protocols describe preparation of the macromolecular sample (i.e., proteins, nucleic acids, macromolecular complexes, and membrane proteins). The preparation and assessment of crystallization trials is then described, along with a protocol for determining whether the crystals obtained are composed of macromolecule or salt. Next, the optimization of crystallization conditions is presented. Finally, protocols that facilitate the growth of larger crystals through seeding are described.
Introduction to NMR of Proteins
Authors: Edwards Andrew J. , Reid David
Published: June 2004

This unit aims to overview the application of NMR spectroscopy to proteins. It is not intended to provide an exhaustive “how to” guide, but rather to give a flavor of both well‐established and emerging NMR techniques used in the elucidation of protein structure. It starts with a brief introduction to the basic principles of NMR and the information provided by this technique, and goes on to discuss the instrumentation involved, spectral assignment methods for small and large proteins, and the utility of other spin active nuclei (e.g.,13C and15N) to aid assignment of the latter.
Introduction to Atomic Force Microscopy (AFM) in Biology
Authors: Goldsbury Claire , Scheuring Simon
Published: June 2004

The atomic force microscope has the unique capability of imaging biological samples with molecular resolution in buffer solution. In addition to providing topographical images of surfaces with nanometer‐ to angstrom‐scale resolution, forces between single molecules and mechanical properties of biological samples can be investigated. Importantly, the measurements are made in buffer solutions, allowing biological samples to stay alive within a physiological‐like environment while temporal changes in structure are measured. This overview provides an introduction to AFM on biological systems and describes specific examples of AFM on proteins. The physical principles of the technique and methodological aspects of its practical use and applications are also described
Raman Spectroscopy of Proteins
Authors: Benevides James M. , Overman Stacy A. , Thomas George J.
Published: June 2004

A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide backbone and its side chains. The spectral positions, intensities, and polarizations of the Raman bands are sensitive to protein secondary, tertiary, and quaternary structures and to side ‐chain orientations and local environments. In favorable cases, the Raman spectrum serves as an empirical signature of protein three‐dimensional structure, intramolecular dynamics, and intermolecular interactions. Here, the strengths of Raman spectroscopy are illustrated by considering recent applications that address (1) subunit folding and recognition in assembly of the icosahedral capsid of bacteriophage P22, (2) orientations of subunit main chains and side chains in native filamentous viruses, (3) roles of cysteine hydrogen bonding in the folding, assembly, and function of virus structural proteins, and (4) structural determinants of protein/DNA recognition in gene regulatory complexes. Conventional Raman, UV‐resonance Raman, and polarized Raman techniques are surveyed.
Synthesis of Multiple Peptides on Plastic Pins
Authors: Rodda Stuart J.
Published: June 2004

This 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

The 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.
Analysis of Protein‐Protein Interactions
Authors: Brent Roger
Published: June 2004

This overview defines some of the terminology used in the study of protein‐protein interactions and also describes the basis of equilibrium kinetic assays (i.e., defining necessary assumptions, terms and equations).
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

The 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

Interaction 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

Arrays 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

The 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

The 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

The 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

Metalloproteases (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

The 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

The 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

The 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

One 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

Current 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

This 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.
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Semba Kentaro
Published: June 2004

In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the scientist. This appendix outlines a few such considerations relevant to the isotopes most frequently used in biological research.
Silanizing Glassware
Authors: Seed Brian
Published: June 2004

Untreated glass contains silicate and silanol groups that can act as ion‐exchange and nucleophilic centers. To mask these groups and decrease the hydrophilicity of the surface, various reactive silanes are frequently used to coat the glass surface. This appendix describes a procedure for silanizing glassware.
Statistics: Detecting Differences Among Groups
Authors: Robakiewicz Phil , Ryder Elizabeth F.
Published: June 2004

Some 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

A 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

Southern 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

Restriction 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

The 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

Neuropeptide 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

This 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

This 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

This 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

Medium 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

This 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.
Angiotensin II Receptor Assays
Authors: Wong Pancras C.
Published: June 2004

This unit describes the use of an isolated rabbit thoracic aorta preparation for determining the nature, potency, and selectivity of antagonists to the agonism of angiotensin II at AT1 receptors by measuring their effects on the tension of the tissue.
Neuropeptide Y (NPY) Receptor Assays
Authors: Daniels Alex , Harrington Wallace
Published: June 2004

Neuropeptide 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.
Cysteinyl Leukotriene Receptor Assays
Authors: Muccitelli Roseanna M. , Luttmann Mark A. , Hay Douglas W.P.
Published: June 2004

The cysteinyl leukotrienes (CysLTs), LTC4, LTD4 and LTE4, lipid products derived from arachidonic acid metabolism, have been implicated in the pathophysiology of several inflammatory diseases, in particular, asthma. This unit describes techniques and applications for the measurement of contractile responses to the CysLTs in isolated smooth muscle preparations. The contractions are assessed by standard methods for the isometric measurement of responses (contractile or relaxant) of isolated tissues to exogenous agonists, and a detailed description of the methods employed to assess CysLT‐induced contractions in guinea‐pig trachea is outlined. However, the same general methodology (other than parameters such as dissection for non‐airway tissues) are appropriate for measuring CysLT‐induced contractions in airway preparations from other animals, and in non‐airways tissues (e.g., the gastrointestinal tract) from different species, and also in exploring the relaxant responses to the CysLTs that have been demonstrated in some tissues (e.g., pulmonary vein or artery).
Models of Inflammation: Carrageenan‐Induced Paw Edema in the Rat
Authors: Whiteley Phyllis E. , Dalrymple Stacie A.
Published: June 2004

Paw swelling, or footpad edema, is a convenient method for assessing inflammatory responses to antigenic challenges and irritants. The protocol described in this unit uses carrageenan as the irritant to induce paw edema. Typically, test compounds are assessed for acute anti‐inflammatory activity by examining their ability to reduce or prevent the development of carrageenan‐induced paw swelling. This model has long been used to assess the anti‐inflammatory properties of agents such as nonsteroidal anti‐inflammatory drugs (NSAIDs) that inhibit prostaglandin production.
Models of Inflammation: Adjuvant‐Induced Arthritis in the Rat
Authors: Whiteley Phyllis E. , Dalrymple Stacie A.
Published: June 2004

Injection of adjuvant (Mycobacterium butyricum suspended in mineral oil) into rats produces an immune reaction that characteristically involves inflammatory destruction of cartilage and bone of the distal joints with concomitant swelling of surrounding tissues. Adjuvant‐induced arthritis in rats is commonly used to evaluate compounds that might be of potential use as drugs for treatment of rheumatoid arthritis and other chronic inflammatory conditions. This unit describes a method for inducing arthritis by injecting adjuvant into the tail and evaluating a test compound for the ability to inhibit the inflammatory response.
Models of Urogenital Dysfunction: Urinary Urge Incontinence (UUI) in Anesthetized Dog and Rat Models
Authors: Newgreen Donald , Anderson Charles , Nunn Philip , Carter Anthony , Bushfield Mark
Published: June 2004

This unit describes a model system for the measurement of the effects of compounds on bladder function in anesthetized dog and rat. A protocol is also described for measuring nerve‐stimulated salivary secretion which is particularly relevant when investigating agents that modulate parasympathetic drive (such as muscarinic antagonists), since it allows for the measurement of relative selectivity of bladder function over salivation. This unit describes a model system for the measurement of the effects of compounds on bladder function in anesthetized dog and
Models of Neurological Disease (Alzheimer's Disease): Spatial Discrimination Water Maze Test in Septal‐Lesioned Rats
Authors: Decker Michael W.
Published: June 2004

Models of Alzheimer s disease currently focus on symptomatology, as understanding of the etiology of the disorder is not yet sufficiently advanced to model the disease process in animals. Moreover, currently available treatments are purely palliative in nature. Given that cognitive dysfunction is a central feature of the disease, animal models of cognitive dysfunction are frequently used to evaluate the therapeutic potential of test compounds. This unit presents a rat model in which a brain lesion is produced which disrupts septohippocampal function, thus impairing performance on a spatial learning task (the Morris water maze).