bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorometric Techniques for the Detection of Nitric Oxide and Metabolites
Authors: Wink David A. , Kim Sungmee , Miles Allen , Jourd'heuil David , Grisham Matthew B.
Published: June 2004

Recently fluorometric techniques have been developed to determine NO and S‐nitrosothiols with improved sensitivity over spectrophotometric techniques based on similar chemistry. This unit contains protocols for detection and quantification of NO from chemical reactions and from cellular systems, and of S‐nitrosothiols in chemical, biochemical, and cellular experiments.
Immunocytochemical Analysis of Cyclic Nucleotides
Authors: deVente J. , Steinbusch H.W.M.
Published: June 2004

Visualization of second messenger molecules is a powerful approach to study the role of second messengers in relation to the (sub)cellular localization and/or kinetics of the second messenger response. This unit describes the appropriate fixation procedures for the cyclic nucleotides cAMP and cGMP and the use of specific antibodies to localize these molecules.
Overview of Neurotoxicology
Authors: Tilson Hugh.A.
Published: June 2004

Environmental exposure to chemicals such as lead, mercury, organic solvents, pesticides, as well as naturally occurring compounds have neurotoxic effects. This unit is an overview of the structure and function of the nervous system, including a discussion of critical issues in defining neurotoxic effects and neurotoxicological risk assessment.
Testing for Organophosphate‐Induced Delayed Polyneuropathy
Authors: Moretto Angelo
Published: June 2004

Organophosphorous compounds may cause two distinct types of toxicity: acute cholinergic toxicity and organophosphate‐induced delayed polyneuropathy (OPIDP). The ability of a compound to cause OPIDP is assessed as described by administering the compound to hens and screening the brain, spinal cord, and peripheral nerves for neuropathy target esterase activity to detect OPIDP and acetylcholinesterase activity to rule out the acute toxicity. This assay can also be used as part of a screen for protective agents.
Risk Assessment and Neurotoxicology
Authors: Slikker William
Published: June 2004

Risk assessment refers to the use of toxicological data to define the relationship between the exposure to an agent and an adverse health outcome. This unit describes the factors involved in the study and evaluation of neurotoxicants to assess the risks associated with exposure.
Neurobehavioral Testing in Humans
Authors: Anger W. Kent , Rohlman Diane S. , Storzbach Daniel
Published: June 2004

The evaluation of the effects of chemical exposures on humans is a worldwide concern, and most chemicals have not been evaluated for neurotoxic effect. Human neurobehavioral research or clinical evaluations of populations exposed to chemicals must be carefully planned and structured. This unit describes the steps required to create such a study, select the appropriate measures, and evaluate the results.
Biochemical Approaches to Studying Neurotoxicity
Authors: Padilla Stephanie
Published: June 2004

This overview provides an introduction to biochemical analysis of toxicant effects on the nervous system. It includes a brief discussion of the salient features of the nervous system and a review of the various approaches used to detect and identify neurotoxicants and their modes of action.
Development of an In Vitro Blood‐Brain Barrier
Authors: Janigro Damir , Stanness Kathe A. , Soderland Carl , Grant Gerald A.
Published: June 2004

The blood‐brain barrier represents a significant protective barrier for the passage of pathogens and toxicants, but it is difficult to study in vivo. This unit describes an in vitro system that can be used as a model for the blood‐brain barrier to study its functions in an accessible format.
Isolation of Neonatal Rat Cortical Astrocytes for Primary Cultures
Authors: Allen Jeffrey W. , Mutkus Lysette A. , Aschner Michael
Published: June 2004

There is increasing interest in the role of astrocytes as mediators of neurotoxicity. This unit describes a method for preparing astrocyte cultures of greater than 95% purity by enzymatic dissociation from neonatal rat brain. These preparations have both high yield and high viability.
Analytical Cytology: Applications to Neurotoxicology
Authors: Catlin Michelle C. , Guizzetti Marina , Ponce Rafael A. , Costa Lucio G. , Kavanagh Terrance J.
Published: June 2004

This unit describes methods for analyzing the effects of neurotoxicants on cell cycle regulation by dual parameter flow cytometry and on cell signaling by quantifying intracellular calcium concentrations within individual cells by scanning confocal laser microscopy or using the fluorescent calcium probe fluo‐3.
Estimating Cell Number in the Central Nervous System by Stereological Methods: The Optical Disector and Fractionator
Authors: Charleston Jay S.
Published: June 2004

This unit describes techniques, based on recent advances in stereological methods, to obtain unbiased estimates of total cell or synapse number in discrete structures of the central nervous system. They combine unbiased counting frames, unbiased systematic random sampling, and unbiased estimates of the structure volume to produce the final estimate of number.
Measurement of Glial Fibrillary Acidic Protein
Authors: O'Callaghan James P.
Published: June 2004

Toxicant‐induced injury of the adult or developing central nervous system results in hypertrophy of astrocytes at the site of injury. The hallmark of this response is enhanced expression of the major intermediate filament protein of astrocytes, glial fibrillary acid protein (GFAP). A protocol is provided for assaying GFAP levels in detergent homogenates of brain tissue using a sandwich ELISA.
Aggregating Neural Cell Cultures
Authors: Honegger Paul
Published: June 2004

Aggregating Neural Cell Cultures (Paul Honegger, University of Lausanne, Lausanne, Switzerland). When freshly dissociated embryonic tissues are kept under gyratory agitation, the cells aggregate to form three‐dimensional spheroids in which the cells can migrate and organize themselves, attaining maximal cellular differentiation after weeks of culture. The three‐dimensional architecture of the aggregates permits direct cell‐to‐cell interactions and the formation of a natural cell matrix, which is fundamental to the acquisition of the histotypic properties of the aggregates. This unit describes protocols for preparing forebrain cells from embryonic rodents for aggregating cultures and maintaining these cultures to the differentiated state.
Coculturing Neurons and Glial Cells
Authors: Viviani Barbara
Published: June 2004

The close relationship between neurons, glia, astroglia, and astrocytes plays a relevant role in modulating a neurotoxic effect and propagating the consequent degenerative event in the brain. After exposure to a neurotoxicant, glial cells are often activated to release substances responsible for the propagation of the damage. the production of such mediators is the result of both neural death and communication between microglia and astrocytes. Because they are simple and highly controlled, cocultures of different cells of the nervous system are a valuable tool in the investigations of these complex relationships. This unit describes protocols to set up neuron‐glia and microglia‐astrocyte sandwich cocultures, in which the selected cell populations grow on two different surfaces. This system has the advantage that the two cell populations can be separated at any point in the exposure without disrupting the cells
integrity and organization
Organ Culture of Midfacial Tissue and Secondary Palate
Authors: Abbott Barbara D.
Published: June 2004

Palatal organ culture provides an in vitro model for the study of the formation of the secondary palate, which forms the roof of the mouth in the developing fetus. The protocol describes the steps for culture of the mid‐facial region of the fetal mouse or rat. In culture the secondary palatal shelves proceed through stages of growth, elevation and fusion in a manner analogous to that occurring in utero. This model provides a tool for studies of mechanisms of normal and abnormal palatogenesis and has applications for developmental biology and toxicology.
Small Animal Models of Hemorrhagic Shock–Induced Liver Dysfunction
Authors: Bauer Inge , Bauer Michael
Published: June 2004

Blood loss and ensuing circulatory failure continues to be the leading cause of death between the ages of 1 and 45 in most western countries. Hepatocellular dysfunction is a key feature of the pathophysiological sequelae of trauma/hemorrhage in patients surviving the first hours and hemorrhagic hypotension has been widely used as a model for indirect liver injury in the critically ill. This unit describes a low‐flow ischemia model of the liver due to hemorrhagic hypotension that can be used to produce a reproducible and graded injury to the pericentral region of the acinus, which is critical for metabolism of xenobiotics. This protocol allows for measurement of cardiac output and regional flow.
Guidelines for Mating Rodents
Authors: Tyl Rochelle W.
Published: June 2004

Guidelines for Mating Rodents (Rochelle W. Tyl, Research Triangle Institute, Research Triangle Park, North Carolina). Mating is an important part of assessing the effects of toxicants on the reproductive tract. This unit describes the set up of mating pairs, determining the success of mating, and assessing the various outcomes of a successful mating to evaluate reproductive function.
Histopathology of the Male Reproductive System I: Techniques
Authors: Creasy Dianne M.
Published: June 2004

Histopathology of the Male Reproductive System: Techniques (Diane Creasy, Huntingdon Life Sciences, East Millstone, New Jersey). Fixation and sampling of the male reproductive system for routine histopathological examination requires special procedures including the use of nonroutine fixatives and nonroutine stains. High‐resolution light microscopy and electron microscopy require additional specialized techniques including perfusion fixation and plastic embedding. Aspects of comparative anatomy are also important for ensuring that specific structures within the reproductive tract are sampled adequately in various species. This unit provides methodology and guidance on what structures to sample and how to fix and prepare tissues from the male reproductive tract for routine and special investigations.
Histopathology of the Male Reproductive System II: Interpretation
Authors: Creasy Dianne M.
Published: June 2004

Histopathology of the Male Reproductive System II: Interpretation (Diane Creasy, Huntingdon Life Sciences, East Millstone, New Jersey) Histopathology is acknowledged as the most sensitive endpoint for detecting testicular toxicity. However, identification and interpretation of chemically induced changes in the testis require fundamental knowledge of spermatogenesis, its dynamics and regulation. Changes in the rest of the reproductive tract are also frequently inter‐related, being the result of or cause of disturbance in testicular spermatogenesis. This unit provides practical guidelines on how to evaluate testicular histopathology and how to utilize staging in a qualitative evaluation. It also provides an overview of the most common chemically induced lesions and their potential significance with respect to etiology and functional consequences.
Epididymal Sperm Count
Authors: Wang Yefan
Published: June 2004

Making Epididymal Sperm Counts (Yefan Wang, TherImmune Research Corporation, Gaithersberg, Maryland). Epididymal sperm counts are a widely used, simple and sensitive method of assessing the effects of male reproductive toxicants on the epididymal and/or testicular site of action. After careful dissection of the tissues and further processing, the sperm suspensions are counted using a hemacytometer and analyzed for effect.
Formation and Functions of Protein Sulfenic Acids
Authors: Poole Leslie B.
Published: June 2004

Protein sulfenic acids are generated as reversibly oxidized cysteinyl residues formed upon reaction of thiols with peroxides, nitric oxide, peroxynitrite, and other reactive oxygen or nitrogen species. They can be stabilized within the protein environment, irreversibly oxidized to sulfinic and sulfonic acids by additional oxidant, condensed with protein or exogenous thiol groups to form disulfide bonds, or directly reduced back to thiols. Sulfenic acids in proteins can act as intermediates in redox catalysis or as critical components in cysteine‐dependent redox regulation.
Local Lymph Node Assays
Authors: Dearman Rebecca J. , Kimber Ian
Published: June 2004

The murine local lymph node assay (LLNA) allows identification of chemicals that have the potential to cause skin sensitization and allergic contact dermatitis. In this test, contact allergens are identified as a function of events occurring during the induction phase of skin sensitization; specifically, stimulation of proliferative responses in draining lymph nodes measured as a function of radiolabeled thymidine incorporation in situ. Two methods are presented. In one, lymph nodes are pooled on an experimental group basis, while in the other, lymph nodes of individual animal are pooled, which enables statistical analyses. These methods have been developed for contact sensitization hazard assessment and provide a robust and reliable method as an alternative to guinea pig tests which measure skin sensitizing potential as a function of challenge‐induced skin reactions in previously sensitized animals. It is also possible to use LLNA data for measurement of relative potency in terms of risk assessment.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C.
Published: June 2004

Mammalian cells in culture are a valuable source of experimental material and/or reagents for many toxicological studies. This unit provides basic information on aseptic technique, preparation of medium, subculturing monolayer cells, passaging suspension cells, freezing and storing monolayer and suspension cells, thawing and recovering cultures, and determining viable cell number.
Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Authors: Kramer Martha F. , Coen Donald M.
Published: June 2004

The polymerase chain reaction (PCR) is used to enzymatically amplify small quantities of specific DNA sequences. The reaction must optimized to specifically amplify the sequences and primers of interest; this includes titration of magnesium chloride and selection of enhancing agents, if appropriate, to minimize nonspecific primer target interactions and maximize the specificity, sensitivity, and yield.
Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
Authors: Voytas Daniel , Ke Ning
Published: June 2004

This unit provides procedures for detecting an quantifying radiolabeled proteins in SDS‐polyacrylamide gels or blots. Autoradiography requires fixed, dried gels before exposure to X‐ray film. The results may be enhanced by using intensifying screens and/or preflashing the film. Alternatively fluorography with organic scintillant‐impregnated gels may be used to enhance signal. The autoradiogram may be quantified by densitometry. Phosphorimaging is a useful alternative to film exposure.
Northern Blot Analysis of RNA
Authors: Bergeron Marcelle , Honkaniemi Jari , Sharp Frank R.
Published: June 2004

This unit describes the processes of extracting RNA from tissues and analyzing it by northern blot hybridization to probe the expression of a particular gene at the transcriptional level.
Spectrophotometric and Colorimetric Determination of Protein Concentration
Authors: Simonian(spectrophotometric methods) Michael H. , Smith(colorimetric methods) John A.
Published: June 2004

Spectrophotometric and Colorimetric Determination of Protein Concentration (Michael H. Simonian, Beckman‐Coulter, Inc., Fullerton, California, and John A. Smith, University of Alabama at Birmingham, Birmingham, Alabama). Measuring absorbance at 280 nm (A280) is one of the oldest methods for determining protein concentration. This method is still widely used because it is simple and does not require incubating the sample with exogenous chromophores. Accordingly, the quantitation of proteins by peptide bond absorption at 205 nm (A205) is more universally applicable. Furthermore, the absorptivity for a given protein at 205 nm is several‐fold greater than that at 280 nm. Thus, lower concentrations of protein can be quantitated with the A205 method. The disadvantage of this method is that some buffers and other components absorb at 205 nm. The aromatic amino acids also exhibit fluorescence emissions when excited by light in the UV range. Measurement of intrinsic fluorescence by aromatic amino acids is primarily used to obtain qualitative information. However, with a protein standard whose aromatic amino acid content is similar to that of the sample, intrinsic fluorescence can be used for quantitation. The most frequently employed colorimetric methods for determining protein concentration are those of Bradford and Lowry.
Dialysis and Concentration of Protein Solutions
Authors: Andrew Sarah M. , Titus Julie A. , Zumstein Louis
Published: June 2004

Conventional dialysis separates small molecules from large molecules by allowing diffusion of only the small molecules through selectively permeable membranes. Dialysis is usually used to change the salt (small‐molecule) composition of a macromolecule‐containing solution. The solution to be dialyzed is placed in a sealed dialysis membrane and immersed in a selected buffer; small solute molecules then equilibrate between the sample and the dialysate. Concomitant with the movement of small solutes across the membrane, however, is the movement of solvent in the opposite direction. There are several simple and relatively inexpensive methods for concentrating protein solutions. Dialysis against Aquacide 11A (Calbiochem), which removes water through the dialysis tubing, may be used. After concentration, the solution must be redialyzed into the appropriate buffer. Another method is to use Immersible‐CX Ultrafilters (Millipore) which, when connected to a vacuum, remove everything below their molecular weight cutoff (MWCO). Alternatively, centrifugal concentrators, which are operated with the aid of ordinary laboratory centrifuges may be used.
Hydrophobicity Profiles for Protein Sequence Analysis
Authors: Krystek Stanley R. , Metzler William J. , Novotny Jiri
Published: June 2004

Hydrophobic interactions are a major force in protein folding and numerous hydropathy scales have been developed to quantify the relative hydrophobicity of the amino acids. Hydropathy profiles can be used to examine the surface features of proteins in order to generate hypotheses that can be confirmed experimentally. This unit describes the application of hydrophobicity plots to typical problems and provides suggested uses for a few selected scales.
Protein Tertiary Structure Prediction
Authors: Xu Dong , Xu Ying
Published: June 2004

This unit addresses how to predict the tertiary structure of a protein from its amino acid sequence using computational methods. Three types of prediction methods‐‐homology modeling, fold recognition, and ab initio prediction‐‐are introduced.
Comparative Protein Structure Prediction
Authors: Marti‐Renom Marc A. , Yerkovich Bozidar , Sali Andrej
Published: June 2004

Functional characterization of a protein sequence is one of the most frequent problems in biology. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3‐D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3‐D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target‐template alignment, model building, and model evaluation. Comparative modeling remains the only method to reliably predict the 3‐D structure of a protein with an accuracy comparable to a low‐resolution experimentally determined structure. This overview unit describes generic considerations for all four steps of comparative modeling, typical modeling errors, and applications of comparative protein structure models.
Spectrophotometric Determination of Protein Concentration
Authors: Grimsley Gerald R. , Pace C. Nick
Published: June 2004

The concentration of a purified protein in solution is most conveniently and accurately measured using absorbance spectroscopy. The absorbance,A, is a linear function of the molar concentration,C, according to the Beer‐Lambert law:A= ɛ ×l×c, where e is the molar absorption coefficient andlis the cell path length. This unit provides protocols for calculation of ɛ for a folded or unfolded protein, making use of the average ɛ values for the three contributing chromophores in proteins (the side chains of Trp, Tyr, and Cys). A basic protocol describes how to measure the concentration of a protein using the calculated ɛ and the Beer‐Lambert law. A sensitive method is provided for measuring the concentration of proteins that contain few if any tryptophan or tyrosine residues, and a simple method is provided for estimating total protein concentration in crude extracts.
Assays for Total Protein
Authors: Lovrien Rex , Matulis Daumantas
Published: June 2004

This unit describes three copper‐based assays to quantitate total protein: the biuret method, a variation of the Lowry method (Hartree‐Lowry method), and the bicinchinonic acid (BCA) assay. Acid hydrolysis of a protein is coupled with ninhydrin detection to quantitate amino acid content of a sample. Ultraviolet spectrophotometry is used to measure total protein and evaluate samples for the presence of contaminants. The Coomassie dye binding, or Bradford, assay is a quite simple assay and frequently is quite sensitive, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acid pH. Finally, dry weight measurement is used to quantitate pure protein. Support protocols describe a technique for heat sealing glass tubes for acid hydrolysis, sample dialysis in polyacrylamide gel wells to remove low‐molecular‐weight contaminants, and TCA precipitation to precipitate and concentrate proteins and remove low‐molecular‐weight contaminants. In performing total protein assays, there are five issues of concern: (1) sensitivity and technique of the method, (2) clear definition of units, (3) interfering compounds, (4) removal of interfering substances before assaying samples, and (5) correlation of information from various techniques. These concerns are discussed at length in the Commentary of this unit.
Biotinylation of Proteins in Solution and on Cell Surfaces
Authors: Luna Elizabeth J.
Published: June 2004

This unit contains three protocols for biotinylation of isolated proteins: attaching biotin to primary amines (e.g., amino groups of lysyl residues); attaching biotin to sulfhydryls (i.e., thiol groups of cysteinyl residues); and attaching biotin to carbohydrate residues on proteins. As biotinylation of lysyl and cysteinyl residues may alter protein function, modification of protein carbohydrates, which is usually innocuous, may be preferable if intact protein function is required (e.g., for activity assays or affinity purification). Biotinylation of cysteinyl thiols requires that disulfide bonds in isolated proteins be reduced before labeling. Biotinylation of surface proteins on living cells is also described using mild reaction conditions. Finally, this unit includes a brief description of methods for detecting biotinylated proteins.
Metabolic Labeling with Amino Acids
Authors: Bonifacino Juan S.
Published: June 2004

Metabolic labeling techniques are used to study biosynthesis, processing, intracellular transport, secretion, degradation, and physical‐chemical properties of proteins. This unit focuses on pulse‐labeling and pulse‐chase experiments done with [35S]‐methionine, but gives directions for labeling with other radiolabeled amino acids, and also offers guidance for the safe use and handling of 35S‐labeled compounds.
Solid‐Phase Profiling of Proteins
Authors: Leung Hon‐chiu Eastwood , Leung Sau‐mei , Karavanov Alex
Published: June 2004

Protein array technologies refer to any fabrication and use of any arrays containing multiple proteins captured on solid surfaces. This valuable tool is used for high‐throughput protein‐protein interaction studies, protein marker discovery and other applications. This unit provides basic protocols for biomarker discovery and protein‐protein interactions. Different kinds of recently emerged protein array technologies and related detection methods are reviewed.
Overview of Cell Fractionation
Authors: Castle J. David
Published: June 2004

This discussion unit describes the most common methods for cell fractionation which provides the essential ingredients for the increasing number of cell‐free assays now being used in test‐tube reconstructions of complex cellular events involving intercompartmental interactions. Gel filtration separates on the basis of size, centrifugation separates on the basis of size and density, and electrophoresis separates on the basis of surface charge density. Centrifugation is the most widely used procedure in cell fractionation and is the only approach commonly used to separate crude tissue homogenates (often having quite large volumes) into subfractions as starting material for more refined purification procedures. Therefore, this overview focuses primarily on fractionation of organelles by centrifugation.
Desalting, Concentration, and Buffer Exchange by Dialysis and Ultrafiltration
Authors: Phillips Allen T.
Published: June 2004

This unit includes a variety of dialysis and ultrafiltration techniques that can be used for desalting, concentration, or buffer exchange. Standard dialysis by diffusion across cellulose tubing is described as a technique used for desalting or buffer exchange. Ultrafiltration under pressure can be used either for concentrating protein or, where the sample volume is replenished with a desired buffer, for desalting/buffer exchange (i.e., diafiltration). A variation is ultrafiltration by tangential flow, which serves the same purposes as ultrafiltration under pressure but is better suited to handling large volumes of solution. Yet another variation is an ultrafiltration technique employing centrifugal microconcentrators, which allow concentration of very small volumes of solution.
Selective Precipitation of Proteins
Authors: Lovrien Rex E. , Matulis Daumantas
Published: June 2004

Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described.
Long‐Term Storage of Proteins
Authors: Carpenter John. F. , Manning Mark C. , Randolph Theodore W.
Published: June 2004

This unit provides a summary of some of the issues that researchers face when attempting to store purified proteins. It briefly explains the stresses that induce protein aggregation the major causes for chemical degradation. It also discusses how to use various storage strategies to increase the long‐term stability of proteins. When appropriate it points out critical mistakes to avoid. This unit provides a summary of some of the issues that researchers face when attempting to store purified proteins.
Production of Recombinant Proteins in Escherichia coli
Authors: LaVallie Edward R.
Published: June 2004

E. coli is the expression system of choice and a substantial body of literature has accumulated on the successful expression of foreign genes in this host. Several problems with protein expression in E. coli have been encountered, and many have been ultimately solved. This unit describes methods that have been developed for production of recombinant proteins in E. coli and potential pitfalls that may be encountered.
Protein Expression in the Baculovirus System
Authors: Bernard Alain , Payton Mark , Radford Kathryn R.
Published: June 2004

Insect cell‐recombinant baculovirus co‐cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post‐translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large‐scale production of recombinant proteins in the baculovirus expression system. A method for large‐scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end‐point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small‐scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS‐PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed‐batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided.
Overview of the Vaccinia Virus Expression System
Authors: Moss Bernard , Earl Patricia L.
Published: June 2004

The vaccinia virus expression system differs from others in that transcription occurs in the cytoplasm of mammalian cells rather than in the nucleus. As a vector, vaccinia virus has a number of useful characteristics, including a capacity that permits cloning large fragments of foreign DNA (20+ kbp) with retention of infectivity, a wide host range, a relatively high level of protein synthesis, and “appropriate” transport, secretion, processing, and posttranslational modifications as dictated by the primary structure of the expressed protein and the cell type used. This overview discusses the life cycle of the vaccinia virus along with effects of vaccinia infection. The vaccinia vector expression system is described along with specific steps for expressing genes using these vectors. Important safety considerations are also presented.
Gene Expression Using the Vaccinia Virus/ T7 RNA Polymerase Hybrid System
Authors: Elroy‐Stein Orna , Moss Bernard
Published: June 2004

This unit describes a transient cytoplasmic expression system that relies on the synthesis of the bacteriophage T7 RNA polymerase in the cytoplasm of mammalian cells. A gene of interest is inserted into a plasmid such that it comes under the control of the T7 RNA polymerase promoter (pT7). Using liposome‐mediated transfection, this recombinant plasmid is introduced into the cytoplasm of cells infected with vTF7‐3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the gene of interest is transcribed with high efficiency by T7 RNA polymerase. For large‐scale work, protocols are provided for insertion of the pT7‐regulated gene into a second recombinant vaccinia virus by homologous recombination and subsequent coinfection with vTF7‐3 into cells grown in suspension or for direct transfection into OST7‐1 cells (a stable cell line that constitutively expresses the T7 RNA polymerase). Expressed protein is then analyzed by pulse‐labeling and purified. One new development to this vaccinia virus/T7 RNA polymerase hybrid expression system described here is the VOTE inducible expression system, which eliminates the need to use two recombinant viruses or a special cell line.
Overview of the Purification of Recombinant Proteins Produced inEscherichia coli
Authors: Wingfield Paul T.
Published: June 2004

The updated version of this unit presents an overview of recombinant protein purification with special emphasis on proteins expressed in E. coli. The first section deals with information pertinent to protein purification that can be derived from translation of the cDNA sequence. This is followed by a discussion of common problems associated with bacterial protein expression. A flow chart summarizes approaches for establishing solubility and localization of bacterially produced proteins. Purification strategies for both soluble and insoluble proteins are also reviewed. A section on glycoproteins produced in bacteria in the nonglycosylated state is included to emphasize that, although they may not be useful for in vivo studies, such proteins are well suited for structural studies. Finally, protein handling, scale and aims of purification, and specialized equipment needed for recombinant protein purification and characterization are discussed. The methodologies and approaches described here are essentially suitable for laboratory‐scale operations.
Overview of Protein Folding
Authors: Pain Roger H.
Published: June 2004

This overview discusses aspects of protein folding including aggregation, folding pathways, disulfide bonds, stabilization of the native functional state, and approaches for achieving an acceptable rate of protein folding starting with a denatured polypeptide.
Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli
Authors: Wingfield Paul T. , Palmer Ira , Liang Shu‐Mei
Published: June 2004

Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed and the are aggregates, (2) the cell wall and outer membrane components of the aggregates are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidineHCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an “oxido‐shuffling” buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein which is then partially purified by gel filtration before folding, and then the protein is folded and oxidized by simple dialyzed against water. A is included for rapidly determining the amount of folded protein that contains the correct disulfide linkage pattern. Finally, folding and purification of a fusion protein is described using metal‐chelate affinity chromatography.
Analytical Ultracentrifugation
Authors: Laue Thomas M.
Published: June 2004

Analytical ultracentrifugation is one of the most powerful, though as yet underexploited, techniques available to molecular biology and biochemistry. This overview describes applications for analytical ultracentrifugation along with important considerations relating to experimental design.
Determining the CD Spectrum of a Protein
Authors: Pain Roger
Published: June 2004

This unit describes the theory behind circular dichroism (CD) and deals with considerations regarding instrumentation and reagents for CD spectrometry. A protocol is provided that outlines the steps in recording a CD spectrum and two support protocols explain the interpretation of near‐UV and far‐UV CD spectra.
Light Scattering
Authors: Harding Stephen E. , Jumel Kornelia
Published: June 2004

Light scattering methods can provide information about the native molecular weight, oligomeric composition, and gross conformation of a protein in solution. These methods are particularly well suited for studying large oligomeric systems or glycoproteins and can be used to characterize much larger structures involving protein such as viruses and even bacterial spores. This overview discusses theory and application of both static and dynamic light scattering analysis of protein solutions.
Rapid Screening of E. coli Extracts by Heteronuclear NMR
Authors: Gronenborn Angela M.
Published: June 2004

Assessing whether a protein or protein complex is amenable to structural analysis is an important component in the structural genomics effort. In particular, if complete sets of structures for entire genomes are to be obtained within a reasonable time frame, high throughput methodologies for all steps along the way have to be developed. These days, cloning and expression systems are highly optimized and a variety of commercially available vectors can be used. However, heterologous proteins or protein domains expressed in bacteria may not be soluble or correctly folded, necessitating intricate solubilization and refolding schemes prior to structural or functional studies. NMR spectroscopy is an important tool for assessing the solubility, stability, and structural integrity of a gene product. It allows efficient evaluation of many variations of polypeptide length and sequence (without time‐ and labor‐intensive purification/refolding procedures) using 1H‐15N‐HSQC spectroscopy of samples of 15N‐labeled proteins directly from crude E. coli extracts. In addition to screening for particular properties of the expressed protein alone, it is also possible to map intermolecular interactions such as ligand binding. In this unit, the basic methodology for bacterial growth, isotope labeling, and spectroscopic evaluation of the protein structure is provided.
Gel‐Filtration Chromatography
Authors: Hagel Lars
Published: June 2004

Three basic protocols are provided in this unit, corresponding to the major applications of gel filtration. One method describes desalting or group separation, in which the target protein and contaminating solutes differ substantially in molecular size. Another is used for protein fractionation, application is in characterizing the molecular dimensions of proteins. A is also provided for calibrating gel filtration columns to be used in estimating molecular size.
HPLC of Peptides and Proteins
Authors: Boysen Reinhard I. , Hearn Milton T.W.
Published: June 2004

High‐performance liquid chromatography (HPLC) is an essential tool for the purification and characterization of biomacromolecules. This unit presents a thorough discussion of the eight types of HPLC currently used, highlighting equipment and start‐up procedures, recommendations for running each type of experiment, and theoretical considerations for the separation of peptides and proteins. This is an excellent primer for HPLC users.
Affinity Purification of Natural Ligands
Authors: Scouten William H.
Published: June 2004

Immobilization of proteins, nucleic acids, and other “bioligands” requires selection of the proper bioligand for the specific application. This decision influences all others: the matrix is chosen and activated by the method that is most appropriate for this specific application and the ligand is coupled under conditions dictated by the activation method and the nature of the ligand (e.g., is it a labile protein or a sturdy enzyme cofactor?). There are many matrices and activation and coupling methods, and new ones are constantly being developed. This unit provides three protocols for activating and coupling proteins and other nucleophilic ligands to beaded agarose gel: cyanogen bromide, p‐nitrophenyl chloroformate, and tresyl chloride.
Purification of Sequence‐Specific DNA‐Binding Proteins by Affinity Chromatography
Authors: Kerrigan Leslie A. , Kadonaga James T.
Published: June 2004

The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. Preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support, is described, and an alternate protocol provides a method to couple DNA to commercially available CNBr‐activated Sepharose. A method for purification of crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin is also provided. A detailed protocol for the actual affinity chromatography procedure is described and a support protocol allows the investigator to determine the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.
Purification of DNA‐Binding Proteins Using Biotin/Streptavidin Affinity Systems
Authors: Chodosh Lewis A. , Buratowski Stephen
Published: June 2004

This unit presents purification protocols that exploit the tight and essentially irreversible complex that biotin forms with streptavidin. A DNA fragment containing a high‐affinity binding site for the protein of interest is prepared and a molecule of biotinylated nucleotide is incorporated into one of the ends of the DNA fragment. The protein of interest is allowed to bind to the high‐affinity recognition site present in the biotinylated fragment. The tetrameric protein streptavidin is then bound to the biotinylated end of the DNA fragment. Next, the protein/biotinylated fragment/streptavidin ternary complex is efficiently removed by adsorption onto a biotin‐containing resin. Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin‐containing resin. Proteins remaining in the supernatant are washed away under conditions that maximize the stability of the DNA‐protein complex. Finally, the protein of interest is eluted from the resin with a high‐salt buffer. Both batch and column formats are presented, as is a protocol for the use of streptavidin‐agarose. A support protocol describes a mobility shift assay for detecting sequence‐specific DNA‐binding proteins.
One‐Dimensional SDS Gel Electrophoresis of Proteins
Authors: Gallagher Sean R.
Published: June 2004

Electrophoresis is used to separate complex mixtures of proteins, to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in the gel matrix; pore size decreases with higher acrylamide concentrations. The combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit contains protocols that gives the standard Laemmli method for discontinuous gel electrophoresis under denaturing conditions, and the standard method for full‐size gels is adapted for the minigel format. Minigels provide rapid separation but give lower resolution. Several alternate protocols are provided for specific applications, including electrophoresis of peptides and small proteins, continuous SDS‐PAGE, ultrathin gels, multiple single‐concentration gels, gradient gels, multiple gradient gels, and multiple gradient minigels.
Two‐Dimensional Gel Electrophoresis
Authors: Harper Sandra , Mozdzanowski Jacek , Speicher David
Published: June 2004

Two‐dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures. Variations of the most common two‐dimensional technique are described in this unit, namely isoelectrofocusing (IEF) and SDS‐PAGE. This unit also includes support protocols describing pI standards and pH profile measurements, casting Immobiline gels, preparation of tissue culture cells and solid tissues for isoelectricfocusing, preparation of molecular weight standards for two‐dimensional gels, and two‐dimensional protein databases.
Protein Detection in Gels Using Fixation
Authors: Echan Lynn A. , Speicher David W.
Published: June 2004

This unit has been recently updated to include information on preformulated gel stains as well as new protocols for Sypro Ruby and silver staining, and gel imaging methodology. Other previously published protocols are also provided for both rapid and acid‐based Coomassie blue staining, and alternate methods for silver staining (i.e., nonammoniacal silver staining, rapid silver staining, and an enhanced‐background, two‐stage method).
Protein Detection in Gels Without Fixation
Authors: Ursitti Jeanine A. , DeSilva Tara M. , Speicher David W.
Published: June 2004

Fixatives remove SDS from gels, causing the protein to become entwined in the gel matrix; subsequent extraction from the gel is usually not practical without partial cleavage of the protein or dissolution of the gel matrix. In this unit, methods for the detection of proteins without fixation are described. In general, the sensitivity of the methods is lower than that of protein detection with fixation. Sensitivity is dependent on the volume of the protein band within the gel; hence, sensitivity is the highest for sharp, narrow bands. Techniques described in this unit include protein detection in gels by SDS precipitation, preparation of contact blots, and use of the fluorescent labels IAEDANS and fluorescamine to detect proteins in gels. Several additional methods, including the use of tryptophan fluorescence, guide strips, and minimal protein staining, are discussed in the Commentary.
Capillary Electrophoresis of Proteins and Peptides
Authors: Burgi Dean , Smith Alan J.
Published: June 2004

Capillary electrophoresis (CE) is a high‐resolution technique for the separation of a wide variety of molecules of biological interest such as metabolites, drugs, amino acids, nucleic acids, and carbohydrates. This unit focuses on the use of CE to separate proteins and peptides based on charge‐to‐mass ratios. Separation of proteins based on their isoelectric points is described along with a protocol for optimizing the separation conditions for a given protein. CE is also used for separations of peptides on an analytical scale and as a micropreparative technique‐‐with either multiple separations that are pooled or a single, larger‐scale separation‐‐for the isolation of peptides from a protease digestion. In most of these examples the same capillary column can be used for all the separations. Only changes in buffer composition, ionic strength, and the presence or absence of additives are required for each specific application.
Immunoblot Detection
Authors: Gallagher Sean
Published: June 2004

Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. Following electrophoretic separation of proteins and transfer from the gel to an appropriate membrane, the immobilized proteins are probed with specific antibodies to identify and quantitate any antigens present. After being probed with primary antibody, the membrane is washed and the antibody‐antigen complexes are identified using horseradish peroxidase (HRPO) or alkaline phosphatase (AP) enzymes coupled to the secondary anti‐immunoglobulin‐G (anti‐IgG) antibody (e.g., goat anti‐rabbit IgG). As described in this unit, the detection enzymes are attached directly or via an avidin‐biotin bridge to the secondary antibody. Chromogenic or luminescent substrates are also described for visualizing the activity.