bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydrophobicity Profiles for Protein Sequence Analysis
Authors: Krystek Stanley R. , Metzler William J. , Novotny Jiri 
Published: June 2004

AbstractHydrophobic 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.
Comparative Protein Structure Prediction
Authors: Marti‐Renom Marc A. , Yerkovich Bozidar , Sali Andrej 
Published: June 2004

AbstractFunctional 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

AbstractThe 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

AbstractThis 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

AbstractThis 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

AbstractMetabolic 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

AbstractProtein 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

AbstractThis 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

AbstractThis 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

AbstractSelective 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

AbstractThis 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

AbstractE. 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

AbstractInsect 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

AbstractThe 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

AbstractThis 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

AbstractThe 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.
Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli
Authors: Wingfield Paul T. , Palmer Ira , Liang Shu‐Mei 
Published: June 2004

AbstractHeterologous 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.
Light Scattering
Authors: Harding Stephen E. , Jumel Kornelia 
Published: June 2004

AbstractLight 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

AbstractAssessing 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

AbstractThree 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

AbstractHigh‐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

AbstractImmobilization 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

AbstractThe 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

AbstractThis 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

AbstractElectrophoresis 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.