bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence analysis of proteins separated by polyacrylamide gel electrophoresis: Towards an integrated protein database
Authors: Aebersold Ruedi , Leavitt John 
Published: May 2005

AbstractImproved technologies or the synergistic use of complementary methods enhance the efficiency of research and permit the exploration of new approaches for the investigation of complex problems. High sensitivity protein sequence analysis and polyacrylamide gel electrophoresis are such complementary methods. Here we summarize the current status of high sensitivity sequence analysis of proteins separated in polyacrylamide gels and discuss strategies by which this technology can cnhance biological research by generating new approaches for the solution of complex, multifacetted problems. Finally, we outline lmminent technological advances in the area of high sensitivity protein sequence analysis and argue that further technological developments will ultimately lead to the generation of an integrated protein database (containing structural and functional as well as physiological information in an easily accessible form) of all the proteins separated by high resolution two‐dimensional gel electrophoresis.
Application of micellar electrokinetic chromatography to pharmaceutical analysis
Authors: Nishi Hiroyuki , Terabe Shigeru 
Published: May 2005

AbstractElectrokinetic chromatography is a new type of analytical separation method which belongs to the group of high performance capillary electrophoretic techniques but whose separation principle is based on that of chromatography. The solute distributes itself between a carrier and the surrounding medium. The carrier, which corresponds to the stationary phase in conventional chromatography, can be transported by electrophoresis with a different velocity from the surrounding medium. The separation is achieved by the differential solute distribution and the differential migration of the carrier. The charged molecules or charged molecular aggregates are employed as the carrier. Various kinds of carriers are available for electrokinetic chromatography along with different partition mechanisms. Among them, micellar electrokinetic chromatography, which employs an ionic micelle as a carrier, has become the most popular method because of its unique and attractive characteristics as well as the separating capability of electrically neutral or nonionic solutes in comparison with capillary zone electrophoresis. The present paper describes the principle, separation characteristics and its application to the analysis of pharmaceuticals.
The separation and detection of alkaline oligoclonal IgG bands in cerebrospinal fluid using immobilised pH gradients
Authors: Cowdrey Geoffrey , Gould Barry , Rees John , Firth Gary 
Published: May 2005

AbstractA method is described for the separation and detection of highly alkaline IgG bands in unconcentrated cerebrospinal fluid (CSF). These bands are frequently found in the cerebrospinal fluid of patients with inflammatory diseases of the central nervous system, particularly in the case of multiple sclerosis, and their detection is an important aid in clinical diagnosis. An isoelectric focusing technique using an immobilised pH gradient in polyacrylamide gel has been developed over the pH range 7–10, producing a linear and stable pH gradient with excellent resolution. After electrofocusing, the protein patterns were blotted onto polyvinylidene difluoride membranes and visualised using anti‐human IgG followed by an enzyme‐labelled second antibody. Blotting could be carried out by capillary diffusion for up to 16 h duration without any loss in resolution. Using this method, highly alkaline intrathecal IgG bands were found in the cerebrospinal fluid of all of the 14 multiple sclerosis patients. There were also 2 patients with alkaline IgG bands in their cerebrospinal fluid who were not diagnosed as multiple sclerosis. By contrast, no alkaline IgG bands with an isoelectric point (pI) greater than 8.6 were found in any of the serum samples studied (n = 50) from patients with various neurological disorders including multiple sclerosis.
Inbred testing of tomato (Lycopersicon esculentum L.) F1 varieties by ultrathin‐layer isoelectric focusing of seed protein
Authors: Van Den Berg Bartel M. 
Published: May 2005

AbstractTo test seed lots of tomato F1 hybrid varieties for the presence of undesirable inbred seeds by electrophoresis, a method has been developed on the basis of ultrathin‐layer isoelectric focusing. The method is based on the genetic variation of the seed protein PRS‐1 which could be visualized by isoelectric focusing of a 5 mm NaCl‐soluble seed protein extract in a pH 6–9 gel followed by protein staining. Two genetic variants of the PRS‐1 protein, PRS‐1+ and PRS‐11, were found among open‐pollinated varieties, as well as among F1 hybrid varieties. The isoelectric points (pI) of the PRS‐1 proteins are 7.1 and 6.1 for PRS‐1+ and PRS‐11, respectively. The PRS‐1 protein is unique to seed tissue and is located primarily in the embryo. A genetic 1:2:1 segregation of the gene Prs‐1 among several F2 populations shows monogenic inheritance. Analysis of commercial F1 hybrid varieties from several seed companies indicated that the Prs‐11 allele, in contrast to the Prs‐1+ allele, is primarily present in gene pools of „Moneymaker type”︁ tomatoes. The described method is generally applicable to all tomato F1 varieties that are heterozygous for the gene Prs‐1. With the described method one person can routinely analyze more than 768 seeds per day.
Preparative free‐flow isoelectric focusing: Modeling and experiments
Authors: Poux Martine , Bertrand Joël 
Published: May 2005

AbstractFree‐flow isoelectric focusing was adapted to preparative scale separations and chemical engineering methods were used to describe the main mechanisms operating in the apparatus. A mixture of human serum albumin (pI 4.6) and β‐lactoglobulin (pI 5.22) was separated in pH gradients, generated with carrier ampholytes of different origin and covering the pH ranges 4‐6.5, 3.5‐5, 4‐5.5 and 4.5‐5.0. Best results were obtained in the pH 4‐5.5 range. The experimental results have validated the results obtained with a numerical model.
Free‐flow electrophoresis for the purification of proteins: II. Isoelectric focusing and field step electrophoresis
Authors: Kuhn Reinhard , Hoffstetter‐Kuhn Sabrina , Wagner Horst 
Published: May 2005

AbstractTwo modes of continuous isoelectric focusing are described. The development of a natural pH gradient, consisting of a mixture of three buffer solutions, and the focusing behavior of human serum albumin is investigated. The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of α‐amylase from an E. coli protein extract. Furthermore the principle of field step electrophoresis is presented. The most important factors influencing the efficiency: (i) residence time, (ii) conductivity of the sample and (iii) sample zone width, are discussed. The use of a larger sized device to allow simultaneous multiple injections of the sample demonstrates the feasibility of scaling‐up field step electrophoresis. This approach permits a throughput of about 20 mL sample solution per minute.
Separation of antibiotics by autofocusing
Authors: Sova Otto 
Published: May 2005

AbstractThe autofocusing separation of nine antibiotics was investigated: tetracycline and chloramphenicol in large‐scale batches, and seven others in gram preparation amounts. All except one were found to be hetergeneous in autofocusing, and 2‐4 well‐characterized fractions emerged. These heterogeneities are described and the industrial utilization of the procedure is suggested.
Quantitative exploration of the REF52 protein database: Cluster analysis reveals the major protein expression profiles in responses to growth regulation, serum stimulation, and viral transformation
Authors: Garrels James I. , Franza B. Robert , Chang Cecile , Latter Gerald 
Published: May 2005

AbstractQuantitative protein databases reveal the response of cells to experimental variables, such as exposure to growth factors or transfection with a transforming gene. The nature of the response depends on the type of cell and its internal state at the time of the stimulus. By constructing a protein database to study a given cell line, we can better understand the differentiated state of the cell, the growth regulatory mechanisms it employs, the particular mechanisms it uses to cope with its environment, and the ways these mechanisms may have been compromised through mutation or transformation. The REF52 database is a quantitative database designed to study growth control and transformation in a well‐defined family of normal and transformed rat cell lines. The database, which has been described and analyzed elsewhere (J. I. Garrels and B. R. Franza, J. Biol. Chem. 1989, 264, 5283–5298 and J. I. Garrels and B. R. Franza, J. Biol. Chem. 1989, 264, 5299–5312) is further explored here using cluster analysis. This method reveals the most common protein expression profiles for each series of two‐dimensional gels without requiring any prior hypothesis or queries on the part of the investigator. This study reveals, for each experiment, large and small clusters of protein expression profiles, most of which have readily apparent biological meaning. For example, large clusters of proteins induced or repressed during growth to confluence have been revealed, and several clusters of transformation‐sensitive proteins reveal differential effects of transformation by DNA‐ and RNA‐tumor viruses. This analysis extends our earlier quantitative explorations of the REF52 protein database and helps to show how such a database can be used to provide context and guidance for molecular studies of regulation in a given cell system.
Automated amplification and sequencing of human mitochondrial DNA
Authors: Sullivan Kevin M. , Hopgood Romellé , Lang Brian , Gill Peter 
Published: May 2005

AbstractPart of the human mitochondrial D‐loop region was amplified by two successive rounds of polymerase chain reaction (PCR) amplification. In the second PCR reaction, nested primers were used, of which one contained the M 13–21 universal primer sequence. By using nonequal concentrations of primers in the second amplification, single‐stranded DNA was generated. This was then sequenced directly by the dideoxy chain termination method using dye‐labelled universal sequencing primers in conjunction with a fluorescence‐based DNA sequencer. This enabled a 403‐base‐pair hypervariable segment of the D‐loop region to be readily sequenced in a single reaction. This paper describes a protocol which enables mitochondrial sequence information to be generated rapidly and automatically. It is likely to be of importance in forensic analysis where the DNA is too degraded or of insufficient quantity to be analysed by other techniques.
Structural analysis of recombinant protein prepared by semi‐dry electroblotting after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Fausset Patricia R. , Lu Hsieng S. 
Published: May 2005

AbstractProteins that were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis were electroblotted onto polyvinyliden difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N‐terminal sequence analysis. A semi‐dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis. This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions. N‐Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N‐terminal sequence analysis of the purified electroblotted samples. Consequently, time‐consuming chromatographic procedures can be eliminated. These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.
Computerized methods for analyzing two‐dimensional agarose gel electropherograms
Authors: Aldroubi Akram , Unser Michael , Tietz Dietmar , Trus Benes 
Published: May 2005

AbstractPrevious methods interpret zonal or polydisperse gel patterns of two‐dimensional Serwer‐type gels in terms of size and free mobility (surface net charge density). These two parameters have been determined for each component without quantitatively measuring the abundance of the components. The present study advances these previous methods by determining the relative concentration of each component by computer evaluation of densitometrically analyzed gel patterns. Suitable procedures and their underlying algorithms are presented. The mathematical routines are implemented in a user‐friendly software package, called GelFit and designed for a Macintosh personal computer. The program input sonsists of digitized images of gel staining patterns exemplified by those obtained from electrophoresis of native sub‐cellular‐sized particles. The data are processed through the following steps: (i) Noise reduction and calibration. (ii) Geometrical transformation of the pattern onto a rectangular size/free mobility coordinate system using rationales of the extended Ogston model. (iii) Analysis of the transformed image to determine density maxima, density profiles along iso‐free‐mobility or iso‐size lines, curve fitting of one‐dimensional profiles or two‐dimensional surfaces using Gaussian functions and curve stripping of surfaces to determine the possible number of particle populations.
Analysis of acrylamido‐buffers for isoelectric focusing by capillary zone electrophoresis
Authors: Righetti Pier Giorgio , Ettori Claudia , Chiari Marcella 
Published: May 2005

AbstractImmobilized pH gradients use a series of weak acrylamido acids and bases (Immobiline) to create a pH gradient along the separation axis. These buffers can be degraded in water by two mechanisms: (i) hydrolysis of the amido bond, with generation of free acrylic acid and either an amino acid or a diamine; (ii) autopolymerization to oligomers and/or n‐mers. In order to check for these degradation products, different capillary zone electrophoresis systems for analysis of all Immobilines have been devised. The acidic compounds are resolved in 100 mM acetate, pH 4.0, whereas the alkaline Immobilines are separated in 50 mM phosphate buffer, pH 7.7 (or pH 7.2 for the weaker species). Polymers of alkaline Immobilines are resolved in 50 mM phosphate buffer, pH 2.5, in 1 % Ficoll‐400. All Immobilines are detected underivatized, by their adsorption at 214 or 254 nm. A calibration curve has been constructed for quantification of acrylic acid contamination. As little as 1 mol% of acrylic acid contamination in Immobiline solutions can be detected, with a sensitivity limit below 0.2 mM (at the injection port).
Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 8‐aminonaphthalene‐1,3,6‐trisulphonic acid: Application to the enzymological structural analysis of oligosaccharides
Authors: Jackson Peter , Williams Gwynn R. 
Published: May 2005

AbstractMono‐ and oligosaccharides, each containing a reducing end group, were labeled with the charged fluorophore 8‐aminonaphthalene‐1,3,6‐trisulphonic acid and the resulting derivatives were separated with high reselution by polyacrylamide gel electrophoresis using a method developed recently (P. Jackson, Biochem. J. 1990, 270, 705–713) but with an alternative electrophoretic buffer system. The fluorescent derivatives of glucose and all its straight chain, α 1–4 linked, oligomers from maltose to maltoheptaose were well resolved. Various isomers such as maltose and lactose could be separated, as were maltose and cellobiose and some epimers, for instance glucose and galactose. The method was applied to the analysis of the partial sequential degradation of a complex oligosaccharide with neuraminidase and β‐galactosidase. Gels showing fluorescent saccharide band patterns were recorded in picomolar quantities either photographically or using an imaging system based on a cooled charge‐coupled device.
Searching for genetic markers for hereditary diseases in cattle by means of DNA fingerprinting
Authors: Doff Gaudenz , Glowatzki Marie‐Louise , Gaillard Claude 
Published: May 2005

AbstractLosses caused by inherited diseases affect not only the breeder's income but also the breeding programs themselves. In Switzerland the cardiomyopathy of cattle and the syndrome of arachnomelia and arthrogryposis are currently being investigated in order to find genetic markers for these diseases. It seems that DNA fingerprinting with multilocus probes is the most efficient approach available at present. Microsatellites analyzed with the polymerase chain reaction technique will provide a usable, marker map within the next ten years and will therefore provide a perfect tool to find markers for hereditary diseases.
Hybridization and polymerase chain reaction amplification of simple repeated DNA sequences for the analysis of forensic stains
Authors: Roewer Lutz , Rieß Olaf , Prokop Otto 
Published: May 2005

AbstractWe have evaluated oligonucleotide hybridization and amplification techniques with regard to quantity and quality of genomic DNA that is under investigation in practical forensic case work. In order to obtain sufficient information from analyzing stain material, we use hypervariable simple repeat sequences for individualization, which occur in all eukaryotic genomes. For the analysis of larger amounts of stains (> 500 ng DNA) the multilocus probes (CAC)5/(GTG)5Probes are written in capitals, genomic noncoding sequences in lower case
are superior because of their discrimination potential – provided that the hybridizing DNA is of high molecular weight. The less discriminating probes (CT)8 and (GACA)4 are more sensitive (minimal amount: 100ng DNA) and still informative when the DNA is degraded. To increase the sensitivity of forensic stain analysis in special cases we have used the polymerase chain reaction technique to amplify hypervariable simple (gt)n/(ga)m repeat structures from the intron 2 of HLA‐DRB genes. Largely independent of the starting amount of DNA and independent of the degradation status, we were able to generate discriminating DNA fragments, which can be used to type (i) microstains and (ii) totally degraded material including human mummy DNA.
Chromosomal organization of simple repeated DNA sequences
Authors: Nanda Indrajit , Zischler Hans , Epplen Conny , Guttenbach Martina , Schmid Michael 
Published: May 2005

AbstractStretches of short, simple DNA sequences are widespread in all eukaryote genomes studied so far. Simple sequences are thought to undergo frequent expansion and deletion due to intrinsic genomic mechanisms. Some of the simple sequences were used successfully to detect hypervariable loci in various genomes. Hybridization experiments using synthetic probes not only revealed the informative simple repeats suitable for DNA fingerprinting in a particular specie, but also reflected the wide range of distribution of the simple sequences among eukaryotes. The organization of these simple repetitive sequences at the chromosomal loci was investigated using in situ hybridization with chemically synthesized, pure oligonucleotide probes. Both biotin‐ and digoxigenin‐attached probes detected specific chromosomal sites that are enriched in the respective simple‐repeat blocks. Depending on the organism and probe used, accumulation of simple DNA sequences at individual or multiple sites on the chromosomes of different vertebrates could be demonstrated. The simple repetitive DNA sequences are located in different chromosomal regions (e. g., heterochromatin on the sex chromosomes, nucleolus organizer regions, and R‐band sites), which are constrained considerably during evolution.
Two‐dimensional electrophoresis in small gels for applications in veterinary medicine
Authors: Miller Ingrid , Gemeiner Manfred 
Published: May 2005

AbstractA versatile method for two‐dimensional electrophoresis that can be performed easily, even in small routine laboratories, is described. The procedure combines a first‐dimensional isoelectric focusing run in a PhastSystem with denaturing electrophoresis in a small vertical electrophoresis chamber. The described arrangement of two first‐dimensional gel strips in the second dimension allows direct comparison of two related samples, eliminating most of the artifacts that usually lead to misinterpretations. The presented procedure can be conveniently adapted to the needs of each laboratory; at present, it is being extensively used to establish proteir patterns of healthy individuals of different species, a prerequisite with regard to help and support diagnosis.
Efficient, small scale electroelution of high molecular weight DNA from agarose gels by a miniature vertical electrophoresis cell
Authors: Pascali Vincenzo L. , Pescarmona Marina , Dobosz Marina , d'Aloja Ernesto 
Published: May 2005

AbstractA miniature, vertical electrophoretic system is described that allows quick elution and efficient harvesting of double‐stranded, high molecular weight DNA fragments from agarose gels. A gel slice containing the band of interest is excised from the agarose gel after submarine electrophoresis and placed into an electrophoresis cell, assembled by inserting a truncated micropipette tip into a 2.0 mL polypropylene tube. The tip supports the agarose slice and is connected to dialysis tubing. Platinum wire electrodes are placed over the tip and at the bottom of the test tube. DNA molecules are quickly eluted into the dialysis bag (60 s to 10 min for bands ranging from 2 to 23 kb) and then easily recovered. The method is highly efficient and allows considerable time saving. Of special interest is its applicability to small starting amounts of DNA (<0.5 μg per band). Purity of the DNA is comparable, if higher, to that obtainable by conventional electroelution on dialysis bags and DEAE–cellulose membrane electrophoresis. The system is simple enough to be used as a routine method for eluting multiple bands in short times.