bluuebunny/crossref_metadata_2025_split huggingface dataset:
Subcellular Fractionation of Tissue Culture Cells
Authors: Aniento Fernando , Gruenberg Jean 
Published: June 2004

AbstractCell fractionation techniques include some of the most important and widely used analytical tools in cell and molecular biology, and are essential for the development of cell‐free assays that reconstitute complicated cellular processes. In addition to simple gradient systems, this unit discusses the immuno‐purification of organelles, in particular endosomes. As antigens, purification can be achieved using endogenous or ectopically expressed proteins, provided that appropriate antibodies are available. Alternatively, tagged proteins can be used, when combined with anti‐tag antibodies. Now that sequencing of the genomes of several organisms has been completed, biochemical strategies, and in particular fractionation and in vitro transport assays, are more necessary than ever to study the numerous protein and protein complexes that are being discovered.
Fermentation and Growth ofEscherichia colifor Optimal Protein Production
Authors: Bernard Alain , Payton Mark 
Published: June 2004

AbstractLarge‐scale production of recombinant proteins inEscherichia colirequires growth of cells in fermentors. This unit listsE. colistrains appropriate for use in fermentors, and also discusses important characteristics of fermentation equipment. Production of recombinant proteins in batch fermentations is described, as are variations of fermentation systems that enable continuous growth and protein production in high‐cell‐density, fed‐batch cultures and that permit labeling of recombinant proteins with heavy atom derivatives such as selenomethionine or with stable isotopes such as2H,13C, and15N. Production of labeled proteins facilitates structural studies by X‐ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. The protocols in this unit are designed for expression systems directing intracellular or periplasmic localization of recombinant proteins; however, in the case of extracellular secretion of the desired protein, the culture medium itself, rather than pelleted cells, would be saved, concentrated, and subjected to purification processes. Fermentation experiments require careful monitoring of cell growth and assurance of preinoculation sterility, both which are described here.
Overview of Protein Expression in Saccharomyces cerevisiae
Authors: Strausberg Robert L. , Strausberg Susan L. 
Published: June 2004

AbstractThis overview presents vectors and host strains that are available to direct gene expression in S. cerevisiae, including information on promoters, vector maintenance and copy number, transcription terminators, and selectable markers. Challenges to the expression of foreign proteins are also covered, including attainment of desired production yield, production of protein with appropriate post‐translational modifications, conformation and function, and secretion to the extracellular medium.
Culture of Yeast for the Production of Heterologous Proteins
Authors: Romanos Michael A. , Clare Jeffrey J. , Brown Crawford 
Published: June 2004

AbstractThis unit describes culture of the yeast strainsSaccharomyces cerevisiaeandPichia pastorisfor the production of foreign proteins. The protocols listed here forS. cerevisiaeare for three widely used types of promoter: galactose‐regulated (GAL1, GAL7, GAL10), glucose‐repressible (e.g.,ADH2), and constitutive glycolytic (e.g.,PGKorGAPDH). Minor variations to each can be made depending on the selection system used. The P. pastoris expression system uses integrating vectors with the methanol‐regulatedAOX1promoter andHIS4selection marker; although transformants are stable, they are generally grown in minimal selective medium. Methods are described for small‐scaleS. cerevisiaeandP. pastoriscultures and also for high‐density fermentations with these yeasts. A simple feeding strategy based on calculated feed rates is provided forS. cerevisiaeand yields cell densities of 10 to 30 g/liter. In contrast, with P. pastoris, basic fermenter equipment is used to obtain extremely high‐density cultures (e.g., 130 g/liter). Finally, adescribes small‐scale preparation of protein extracts.
Generation of Recombinant Vaccinia Viruses
Authors: Earl Patricia L. , Moss Bernard , Wyatt Linda S. , Carroll Miles W. 
Published: June 2004

AbstractThis unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid‐transfer vector to generate a recombinant virus. Methods are also presented for purifying vaccinia virus and for isolating viral DNA, which can be used during transfection. Also presented are selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.
Choice of Cellular Protein Expression System
Authors: Gray David , Subramanian Shyam 
Published: June 2004

AbstractRecombinant protein expression has become a standard laboratory tool, and a wide variety of systems and techniques are now in use. Because there are so many systems to choose from, the investigator has to be careful to use the combination that will give the best results for the protein being studied. This overview unit discusses expression and production choices, including post‐translational modifications (e.g., glycosylation, acylation, sulfation, and removal of N‐terminal methionine), in vivo and in vitro folding, and influence of downstream elements on expression.
Determining the Fluorescence Spectrum of a Protein
Authors: Pain Roger H. 
Published: June 2004

AbstractFluorescence spectra of proteins are determined chiefly by the polarity of the environment of the tryptophan and tyrosine residues and by their specific interactions. A thorough consideration of fluorescence spectrometers and their calibration is provided along with important information regarding spectrometer cells, buffers and clarification of samples. Protocols are provided for recording fluorescence spectra and for measuring fluorescence quenching to probe the accessibility of tryptophan residues to small molecules (to yield information about the structural environment of the tryptophan). The technique involves quantifying the decrease in protein fluorescence intensity in the presence of increasing concentrations of quencher, followed by analysis of the data to give details of the interaction of the quencher with the tryptophan residue. Finally, agives details on how to interpret fluorescence spectra.
Overview of Conventional Chromatography
Authors: Williams Alan 
Published: June 2004

AbstractThe key to success in purification of any protein is developing the right separation strategy. This involves selecting and correctly applying a combination of separation techniques, chosen on the basis of information about the target protein and contaminants. There is no single separation technique and no single purification scheme that will allow successful purification of all types of proteins. However, there is a single approach to developing a successful purification strategy. This overview discusses the basic steps that must be taken, including selection of source material, and progression from initial “capture” stage of separation to the final polishing stage. Factors that can affect chromatographic resolution are also covered.
Ion‐Exchange Chromatography
Authors: Williams Alan , Frasca Verna 
Published: June 2004

AbstractThis unit outlines the basic steps in planning and carrying out ion‐exchange chromatography to separate proteins. Protocols describe both batch adsorption and column chromatography in conjunction with either step‐ or linear elution gradients. Support protocols describe (1) pilot experiments to determine initial conditions for batch or column chromatography (i.e., pH required for binding, change in pH or salt concentration required for elution, and available capacity of a medium), (2) calculation of the dynamic capacity of an ion‐exchange column, (3) methods for producing continuous gradients of pH and salt concentration to elute proteins from ion‐exchange columns, (4) regeneration of used ion‐exchange media, and (5) storage of ion‐exchange media.
Hydrophobic‐Interaction Chromatography
Authors: Kennedy Robert M. 
Published: June 2004

AbstractThis unit discusses the important parameters in designing and optimizing a separation by hydrophobic interaction chromatography (HIC), including preparing the sample and choosing a matrix, column, and buffer. Protocols are provided for packing and testing a column; determining binding and elution conditions; eluting the sample; and cleaning, regenerating, and storing HIC columns.
Dye Affinity Chromatography
Authors: Stellwagen Earle 
Published: June 2004

AbstractDye affinity chromatography is a protein purification procedure based on the high affinity of immobilized dyes for the binding sites on many proteins. It is a rapid, inexpensive, and versatile method that is applicable to the purification of crude cellular extracts. This unit presents protocol for the three types of dye affinity chromatography: negative chromatography, positive chromatography, and tandem chromatography. An initial protocol describes a chromatographic procedure in which a small volume of the protein mixture to be purified is applied to a series of miniature columns, each containing a different immobilized dye. Analysis of the flowthrough and bound material allows determination of the optimum dye material for larger‐scale purification. An alternate procedure describes a similar initial selection procedure using centrifugation instead of chromatography. A support protocol describes a simple procedure for immobilization of free dyes.
Immunoprecipitation
Authors: Bonifacino Juan S. , Dell'Angelica Esteban C. , Springer Timothy A. 
Published: June 2004

AbstractImmunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. It is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or density gradient sedimentation. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or intrinsically labeled cells, or in‐vitro‐translated proteins. This unit describes a wide range of immunoprecipitation techniques, using either suspension or adherent cells lysed by various means (e.g., with and without detergent, using glass beads, etc.). Flow charts and figures give the user a clear‐cut explanation of the options for employing the technology.
One‐Dimensional Electrophoresis Using Nondenaturing Conditions
Authors: Gallagher Sean R. 
Published: June 2004

AbstractNondenaturing or “native“ electrophoresis—i.e., electrophoresis in the absence of denaturants such as detergents and urea—is an often‐overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Two protocols are presented in this unit: continuous PAGE, which is highly flexible, permitting cationic and anionic electrophoresis over a full range of pH, and discontinuous PAGE, which is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration.
Capillary Electrophoresis of Peptides and Proteins Using Isoelectric Buffers
Authors: Righetti Pier Giorgio , Bossi Alessandra , Gelfi Cecilia 
Published: June 2004

AbstractCapillary electrophoresis in acidic isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and no interaction of macromolecules with the untreated wall ensues. In addition, because of the low conductivity of quasi‐stationary isoelectric buffers, high voltage gradients can be applied (up to 800 V/cm), permitting fast peptide analysis with a high resolving power and minimal diffusional peak spreading.
Digestion of Proteins in Gels for Sequence Analysis
Authors: Stone Kathryn L. , Williams Kenneth R. 
Published: June 2004

AbstractA high percentage of eukaroytic proteins have blocked amino termini, so it is usually necessary to cleave the protein chemically or enzymatically to obtain the partial sequences needed for cDNA cloning. Because SDS‐PAGE is the current method of choice for the final purification of the >25 pmol amounts of protein that are usually required for internal sequencing, procedures that can be used to digest proteins in situ in SDS‐polyacrylamide gels are often the most useful and have the added benefit of eliminating losses that may occur during blotting. Two strategies have been developed to respond to this need and to deal with the unique problems posed by SDS, which are that SDS inhibits trypsin, one of the enzymes that is most commonly used for internal sequencing studies, and also interferes with reversed‐phase HPLC. In this unit, SDS is removed from the gel prior to enzymatic cleavage by the staining and subsequent washing steps. An acetonitrile wash is performed to remove residual SDS from the protein sample, and Tween 20 is then added back to the sample to maintain the solubility of the denatured protein. In an alternate protocol, the gel slices are washed with ammonium bicarbonate and the protein samples digested in the absence of detergent. Support protocols are provided for determining the amount of protein present in the sample via amino acid analysis, and for reducing and alkylating proteins separated by SDS‐PAGE (which facilitates the identification of cysteine residues during subsequent peptide sequencing reactions).
Chemical Cleavage of Proteins in Solution
Authors: Crimmins Dan L. , Mische Sheenah M. , Denslow Nancy D. 
Published: June 2004

AbstractDescribed in this unit are five basic protocols that are widely used for specific and efficient chemical cleavage of proteins in solution. Cyanogen bromide (CNBr) cleaves at methionine (Met) residues; BNPS‐skatole cleaves at tryptophan (Trp) residues; formic acid cleaves at aspartic acid‐proline (Asp‐Pro) peptide bonds; hydroxylamine cleaves at asparagine‐glycine (Asn‐Gly) peptide bonds, and 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues. Because the above loci are at relatively low abundance in most proteins, digestion with these agents will yield relatively long peptides.
Amino Acid Analysis
Authors: Crabb John W. , West Karen A. , Dodson W. Scott , Hulmes Jeffrey D. 
Published: June 2004

AbstractAmino acid analysis (AAA) is one of the best methods to quantify peptides and proteins. Two general approaches to quantitative AAA exist, namely, classical postcolumn derivatization following ion‐exchange chromatography and precolumn derivatization followed by reversed‐phase HPLC (RP‐HPLC). Excellent instrumentation and several specific methodologies are available for both approaches, and both have advantages and disadvantages. This unit focuses on picomole‐level AAA of peptides and proteins using the most popular precolumn‐derivatization method, namely, phenylthiocarbamyl amino acid analysis (PTC‐AAA). It is directed primarily toward those interested in establishing the technology with a modest budget. PTC derivatization and analysis conditions are described, and support and alternate protocols describe additional techniques necessary or useful for most any AAA method‐‐e.g., sample preparation, hydrolysis, instrument calibration, data interpretation, and analysis of difficult or unusual residues such as cysteine, tryptophan, phosphoamino acids, and hydroxyproline.
Phosphoamino Acid Analysis
Authors: Sefton Bartholomew M. 
Published: June 2004

AbstractIt is often valuable to identify the phosphorylated residue in a protein. This unit presents a protocol for partial acid hydrolysis of proteins phosphorylated at serine, threonine, or tyrosine, followed by two‐dimensional thin‐layer electrophoresis of the labeled phosphoamino acid. Phosphothreonine and phosphotyrosine are more stable to hydrolysis in alkali than are RNA and phosphoserine. Therefore, an alternate procedure using mild alkaline hydrolysis of protein samples to enhance the detection of phosphothreonine and phosphotyrosine is also provided.
Detection of Phosphorylation by Immunological Techniques
Authors: Sefton Bartholomew M. 
Published: June 2004

AbstractPhosphorylation of unlabeled proteins can be detected using immunological or enzymatic techniques. Anti‐phosphotyrosine antibodies are used with immunoblots to detect tyrosine phosphorylation. This unit presents a protocol employing anti‐phosphotyrosine antibodies with detection by either 125I‐labeled protein A or enhanced chemiluminescence (ECL).
Permeabilization Strategies to Study Protein Phosphorylation
Authors: Carter A. Nigel 
Published: June 2004

AbstractThis unit deals with the use of nucleotide triphosphates to label proteins in vitro in permeabilized cells and isolated cellular fractions. Both of these assay formats result in lysates from which the protein of interest may be easily immunoprecipitated; however alternative techniques are described for preparing the final lysate for electrophoretic analysis. A related procedure that does not involve permeabilization is outlined for direct analysis of cytosolic or membrane‐bound kinases. Two different methods for determining the specific radioactivity of 32P‐containing compounds are also included. These experiments generally utilize [γ‐32P]ATP as an exogenously added phosphate donor, although[γ‐32P]GTP can be used in specific cases. The method is very straightforward, although numerous considerations must be made before applying it to each new system.
Overview of Peptide and Protein Analysis by Mass Spectrometry
Authors: Carr Steven A. , Annan Ronald S. 
Published: June 2004

AbstractOne goal of this introductory overview is to try and dispel the still widely held, but now mistaken, belief that mass spectrometry is beyond the technical resources of the typical laboratory or facility engaged in the structural characterization or synthesis of peptides and proteins. Over the last five years, mass spectrometers and associated data systems have become available that can be operated by anyone capable of running a modern amino acid analyzer or Edman sequencer, yet are powerful enough to handle the most demanding analyses that the peptide or protein investigator might require. The remaining goals of this article are to familiarize peptide and protein chemists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide. The emphasis here is on established tools and techniques that can realistically be used for problem solving. As a result, many useful instruments and techniques employed by the trained mass spectroscopist are not discussed here, because they do not satisfy these criteria or are not yet widely available; however, a few of these are briefly mentioned at the end of the unit. The overview concludes with a tutorial discussion on the meaning and practical importance of a number of fundamental experimental and performance‐related issues that have important implications for interpretation and use of mass spectral data, such as mass accuracy and resolution.
Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using Microscale Capillary Liquid Chromatography
Authors: Lee Terry D. , Moore Roger E. , Young Mary K. 
Published: June 2004

AbstractThis unit describes the design and operation of a microscale electrospray (ES) interface suitable for the on‐line liquid chromatography (LC) separation and mass spectrometry (MS) analysis of mixtures of peptides and proteins. The interface utilizes an ES needle packed with reversed‐phase support. Such a design has the advantage of minimizing any void volume between the end of the column and point of electrospray ionization, thus maintaining the integrity of the LC separation and maximizing sensitivity. Here, protocols are presented for construction of an integrated LC column ES needle in‐house, packing the ES needle, and mounting and using the microscale ES LC/MS interface assembly. Various options for low‐flow solvent delivery systems are also discussed.
Principles of Macromolecular X‐Ray Crystallography
Authors: Hickman Alison B. , Davies David R. 
Published: June 2004

AbstractX‐ray crystallography is one of the major tools available for protein structural analysis. This unit provides an introductory review of the principles of X‐ray crystallography that covers how the image is generated and analyzed. The second half of the unit describes strategies for producing crystals of protein, including methods for dealing with proteins that do not easily form crystals.
Probing Protein Structure and Dynamics by Hydrogen Exchange–Mass Spectrometry
Authors: Wang Lintao , Smith David L. 
Published: June 2004

AbstractHydrogen exchange rates at peptide backbone amide linkages of proteins are highly sensitive to changes in protein structure and dynamics. Hydrogen exchange‐mass spectrometry (HX‐MS) can be used to measure hydrogen/deuterium exchange in protein fragments and allows the study of large proteins with high sensitivity and at very low concentrations as well as determination of the intermolecular distribution of deuterium. This unit provides a brief discussion of the interplay between protein dynamics and hydrogen exchange, a detailed prescription for designing and performing typical HX‐MS experiments, and common procedures for processing and interpreting HX‐MS data.
Titration Microcalorimetry
Authors: Doyle Michael L. 
Published: June 2004

AbstractIsothermal titration calorimetry (ITC) is perhaps the most rigorous commercially available method for characterizing protein‐ligand interactions. In this method, interactions are detected by the intrinsic heat (binding enthalpy) change of the reaction. The technique is applicable to native, unmodified proteins in solution. This is important for proteins that lose or change their functional behavior when chemically modified or attached to a surface. ITC is also useful for evaluating qualitative questions such whether a proposed binding interaction occurs at all, or for quantitatively measuring the concentration of functionally active protein. Finally, if executed with proper control experiments, ITC can be a rich source of thermodynamic information about the molecular binding mechanism.
Size‐Exclusion Chromatography with On‐Line Light Scattering
Authors: Arakawa Tsutomu , Wen Jie 
Published: June 2004

AbstractThis unit describes the use of size‐exclusion chromatography with on‐line light scattering, UV absorbance, and refractive index detectors (SEC‐LS/UV/RI) to determine: (a) the molecular weight of simple proteins containing no carbohydrates, (b) the molecular weight of glycoproteins, and (c), most importantly, the molecular weight and stoichiometry of protein‐protein complexes or protein‐carbohydrate complexes. Multiangle light scattering is also discussed.
Serpins (Serine Protease Inhibitors)
Authors: Bauman Susannah J. , Whinna Herbert C. , Church Frank C. 
Published: June 2004

AbstractSerpins are a class of proteins involved in the regulation of serine and other types of proteases. In humans, the majority of serpins regulate the functions of proteases involved in the body's response to injury. This includes roles in coagulation, fibrinolysis, inflammation, wound healing, and tissue repair. Serpins have been implicated in various animal and human pathologies by the loss of a functional serpin gene through deletion or mutation, which results in a defect in functional protein. Examples of sestorically called antithrombin III) are first described. Then, protocols to determine the second‐order rate constant of AT inhibition of thrombin in the absence and presence of heparin are presented. Also provided is a partial list of other serpins and their purification methods.