bluuebunny/crossref_metadata_2025_split huggingface dataset:
Capillary and slab gel electrophoresis profiling of oligosaccharides
Authors: Guttman András , Starr Christopher 
Published: May 2005

AbstractCapillary electrophoresis with laser‐induced fluorescent detection, a one‐dimensional version of the well‐established planar analytical method of polyacrylamide gel electrophoresis, has been proven to be a powerful new microanalytical method for profiling complex carbohydrates. In this paper a comparison is presented between the planar high concentration polyacrylamide gel electrophoresis method and capillary electrophoresis of different carbohydrates with respect to performance and efficiency. N‐Linked oligosaccharides were released from several glycoproteins, including fetuin, human immunodeficiency virus (HIV) envelope recombinant glycoprotein (GP‐120), α1‐acid glycoprotein and ribonuclease B, using recombinant peptide‐N‐glycosidase F (PNGase F). Both separation methods involve labeling of the released carbohydrates at the reducing end with the fluorescent dye, disodium 8‐amino‐1,3,6‐naphthalene trisulfonate (ANTS). Fluorophore labeling was followed by separation of the labeled oligosaccharides either by high concentration polyacrylamide gel electrophoresis or capillary electrophoresis.
Group‐specific component subtyping in semen and seminal stains by conventional isoelectric focusing
Authors: Kido Akira , Kimura Yukiko , Oya Masakazu 
Published: May 2005

AbstractGroup‐specific component (GC) subtyping in semen and seminal stains was carried out using isoelectric focusing in carrier ampholyte‐generated pH gradients and immunoblotting. In serum samples the anodal bands of GC 1F and of GC 1S disappeared by neuraminidase treatment, but in semen samples these bands remained unchanged after such treatment. The GC 2 type in semen exhibited two bands: the main GC 2 band and another fast band which focused at the position of the cathodic band of GC 1F. These seminal GC bands were unaffected by enzyme digestion. Reliable subtyping was possible in seminal stains stored at 4°C for up to 10 weeks, at room temperature for up to 8 weeks, and at 37°C for up to 5 weeks. The GC subtyping by conventional isoelectric focusing after neuraminidase treatment is simple, economical and useful in medicolegal examination of seminal stains.
Two‐dimensional electrophoretic analysis of down‐regulated proteins in human fibroblasts immortalized by treatment with either 4‐nitroquinoline 1‐oxide or 60Co gamma rays
Authors: Kondo Tadashi , Mihara Koichiro , Inoue Yusuke , Iijima Mikio , Namba Masayoshi 
Published: May 2005

AbstractCellular proteins were examined by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) in order to determine the mechanisms of cellular aging and immortalization of human fibroblasts. A total of 10 cell lines were studied, four of which were immortalized. OUMS‐24F and SUSM‐1 cells were immortalized by repeated treatment with 4‐nitroquinoline 1‐oxide (4NQO), KMST‐6 cells were immortalized by intermittent exposure to 60Co‐gamma rays, and IMR‐90/SV40 cells were immortalized with simian virus 40 (SV40). None of these immortalized cells were tumorigenic. Four normal human fibroblast cell lines, OUMS‐24, AD 387, KMS‐6 and IMR‐90, and two human cervical cancer cell lines, HeLa and A‐431, were also studied. Applying 2‐D PAGE, the down‐regulation of the identical proteins was observed in the four immortalized cell lines and in the two human cancer cell lines. Their molecular masses were about 80 kDa and the isoelectric points ranged between 5.5 and 6.3. We could not find any information on our proteins from the recent protein data bases. Further characterization of the proteins indicated that they might be phosphorylated cytoskeletal proteins.
Matrix‐assisted laser‐desorption/ionization mass spectrometric approaches for the identification of gel‐separated proteins in the 5–50 pmol range
Authors: Patterson Scott D. 
Published: May 2005

AbstractThe ability to identify and characterize low picomole quantities of gel‐separated proteins has greatly benefited from recent advancements in mass spectrometric analysis methods, particularly peptide‐mass search routines. We are investigating the use of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS)) to gain as much mass information as possible from a single gel‐separated protein species. This report details results obtained from one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) separations, under nonreducing conditions, of known quantities of three proteins, followed by blotting to Immobilon‐CD. Three methods were used to obtain MALDI‐MS data from a single blotted protein band: (i) direct MALDI‐MS of ∼10% of the band, (ii) cyanogen bromide (CNBr) cleavage of another ∼10% of the band, and (iii) enzymatic (endoproteinase Lys‐C) digestion of the remaining ∼70–80% of the band followed by MALDI‐MS. At the level of 50 pmol of protein loaded onto the gel, data was obtained from all three approaches. At levels down to 5 pmol of protein loaded onto the gel, MALDI‐MS data was obtained from the latter two methods, CNBr and Lys‐C digestions, but not direct MALDI‐MS. Sufficient peptide masses were obtained from the 5 pmol loads to identify two of the three test proteins using four mass search programs. Only limited peptide mass data was obtained from fetuin, a sialylated glycoprotein with six disulfides and no methionines, but it was identified.
Nonreducing two‐dimensional polyacrylamide gel electrophoretic analysis of human colonic proteins
Authors: Reid Gavin E. , Ji Hong , Eddes James S. , Moritz Robert L. , Simpson Richard J. 
Published: May 2005

AbstractImmunochemical detection of proteins with antigenic determinants that are dependent on the native spatial conformation of the protein can often pose problems with conventional two‐dimensional polyacrylamide gel electrophoresis (2‐DE). For example, many antigenic determinants are readily destroyed by reducing agents and/or urea, reagents which are critical components of many of the conventional isoelectric focusing and immobilized‐pH‐gradient (IPG) protocols used in the first electrophoretic dimension. Here we describe the use of commercially available precast 2‐DE gels for performing nonreducing/non‐urea 2‐DE of proteins extracted from the human colon cancer cell line LIM 1215 with 0.3% Triton X‐100 that permit the identification of antigens with conformational determinants by immunoblot analysis. Previous, related studies demonstrated the usefulness of peptide‐mass fingerprinting for identifying 2‐DE resolved proteins. Here we show how partial protein sequence data obtained by rapid peptide mapping, using capillary column liquid chromatography directly coupled with electrospray ionization tandem mass spectrometric methodologies, enhances the usefulness of this approach for identifying incompletely resolved proteins. The nonreducing 2‐DE gel images reported in this study, along with our master 2‐DE gel protein database for both normal human colonic crypts and several colon‐cancer‐derived cell lines, and information regarding microtechniques employed in this laboratory for obtaining structural data on 2‐DE resolved proteins can be accessed over the Internet using World Wide Web (URL address: http://www.ludwig.edu.au).
A Saccharomyces cerevisiae Internet protein resource now available
Authors: Latter Gerald I. , Boutell Thomas , Monardo Patrick J. , Kobayashi Ryuji , Futcher Bruce , McLaughlin Calvin S. , Garrels James I. 
Published: May 2005

AbstractThe QUEST Protein Database Center is now making available two Saccharomyces cerevisiae protein databases via the Internet. The yeast electrophoretic protein database (YEPD) is a database of approximately one hundred protein identifications on two‐dimensional gels. The yeast protein database (YPD) is a database of gene names and properties of over 3500 yeast proteins of known sequence. These databases can be accessed via a World‐Wide Web (WWW) server (http://siva.cshl.org). YPD is available via public ftp (isis.cshl.org) as well, in a spreadsheet format, and in ASCII format. When accessed via WWW, both of these databases have hypertext links to other biological data, such as the SWISS‐PROT protein sequence database and the Saccharomyces Genome Database (SacchDB), and to each other.
Phenotyping of apolipoprotein E using immobilized pH gradient gels for one‐dimensional and two‐dimensional separations
Authors: Golaz Olivier , Sanchez Jean‐Charles , James Richard W. , Hochstrasser Denis F. 
Published: May 2005

AbstractApolipoprotein E (apo E) is a normal component of several classes of plasma lipoproteins. Apo E phenotypes are closely related to total cholesterol, low density lipoprotein (LDL)‐cholesterol and apo B concentration. The apo E 2/2 phenotype is related to the type III hyperlipoproteinemia due to the defective binding of apo E‐2 to the hepatic receptors. The apo E 4/4 phenotype has been reported to be present in most elderly people suffering from the Alzheimer disease, and is associated with increased risk of coronary heart disease and Creutzfeld‐Jakob disease. Therefore, apo E phenotyping is essential. The method described here uses a precast immobilized pH gradient, avoids time‐consuming separation of lipoproteins from plasma, needs no pretreatment with neuraminidase and involves highly sensitive enhanced chemiluminescence for visualization. Therefore it has many advantages over previously published methods.
Two‐dimensional electrophoresis for the study of blood/serum proteins of the otter, an endangered species
Authors: Miller Ingrid , Gutleb Arno C. , Gemeiner Manfred 
Published: May 2005

AbstractStudies on serum/blood samples from European otters (Lutra lutra), a species under protection in most countries, were undertaken. Building of a two‐dimensional electrophoresis (2‐DE) protein identification map was started, identifying serum proteins by pattern comparison with cat or human serum protein maps and by immunoblotting using cross‐reacting antibodies against the respective human serum proteins. The capture of endangered species is very restricted, thus making fresh samples almost unavailable; most investigations therefore had to be performed on samples collected from animals found dead. Patterns obtained from specimens of this source were compared to the established pattern of serum proteins and of blood or liver cells. Some of the major differences observed can be explained by contamination with cellular proteins and/or the onset of lytic processes generating breakdown products, as shown by preliminary in vitro experiments. Better knowledge of “normal” serum protein patterns and interfering products will facilitate interpretation of data collected from samples with a less defined history.
Spermatocytes and round spermatids of rat testis: Protein patterns
Authors: Cossio Gabriela , Sanchez Jean‐Charles , Golaz Olivier , Wettstein Rodolfo , Hochstrasser Denis F. 
Published: May 2005

AbstractSpermatogenesis is a process in the testis that involves meiotic cell division and spermiogenesis. The mechanisms of regulation and its associated proteins are mostly unknown. This publication shows the two‐dimensional (2‐D) gel electrophoresis protein map obtained from rat testis using nonlinear 3.5–10 immobilized pH gradients for the first‐dimensional separation. Eighteen proteins were successfully identified in the SWISS‐PROT protein database using amino acid analysis of proteins recovered from polyvinylidene difluoride (PVDF) membranes and verified for one of them by comparison with Anderson's rat liver reference map. Fourteen new polypeptides were identified and four were previously known. Two of these new proteins were closely related to the spermatogenetic process. T‐complex protein 1 is expressed in large amounts in germ cells. Androgen‐dependent sperm‐coating glycoprotein is secreted by epididymal cells. In order to detect changes in protein expression during meiosis and spermiogenesis, spermatocytes and round spermatid cell populations were purified by centrifugal elutriation and compared. In this way several proteins not found in the spermatocyte 2‐D images could be highlighted. The sperm‐coating glycoprotein was thus shown to be present in large amounts in round spermatids.
Long‐term induction of an aldose reductase protein by basic fibroblast growth factor in rat astrocytes in vitro
Authors: Laeng Pascal , Bouillon Philippe , Taupenot Laurent , Labourdette Gérard 
Published: May 2005

AbstractBasic fibroblast growth factor (bFGF) is known to elicit various developmental‐like effects on astrocytes in vitro, but these effects were studied mainly over short‐term periods. In this work we asked the question whether bFGF could induce long‐term effects on rat astrocytes in culture. This factor was found to induce only a transient mitogenic effect lasting less than 48 h, even when the treatment was carried on for 4 days. By contrast, it induced long‐term effects on the rate of synthesis of several proteins as seen by two‐dimensional polyacrylamide gel electrophoresis after labeling the cells with [35S]methionine. The most upregulated protein was extracted from preparative gels of soluble extracts of cultured bFGF‐treated astrocytes and of normal brain. It was characterized by internal amino acid microsequencing. Two tryptic digest peptides had N‐terminal sequences similar to rat lens aldose reductase. This protein was also expressed in oligodendroglial and neuronal cells in culture, but it was not upregulated by bFGF. Aldose reductase is thought to be involved in a minor pathway of glucose metabolism and in diabetic complications. Its longterm regulation by bFGF will possibly help in the understanding of its actual physiological role.
Programmed cell death in sympathetic neurons: A study by two‐dimensional polyacrylamide gel electrophoresis using computer image analysis
Authors: Amess Bob , Tolkovsky Aviva M. 
Published: May 2005

AbstractThe technique of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) coupled with computer image analysis was used in this study to examine changes in protein expression occurring during the onset of programmed cell death (PCD) in rat sympathetic neurons following withdrawal of nerve growth factor (NGF). Sympathetic neurons from superior cervical ganglia of postnatal day‐one Wistar rats were cultured in the presence of NGF for 24 h and then either maintained in the presence of NGF or deprived of NGF for a period of 8 h. To label the proteins being synthesised, neurons were cultured in the presence of L‐[35S]methionine for a further 2 h under the same conditions but with 3% of the normal methionine concentration. Neuronal proteins were then analysed by 2‐D PAGE using immobilised pH gradient (IPG) gel strips in the first dimension. For the second dimension a custom‐built electrophoresis system capable of running multiple sodium dodecyl sulfate (SDS)‐PAGE slab gels in a vertical configuration, with good temperature control (± 0.7°C) was used and is described in this paper. Proteins resolved on the dried gels were visualised using storage phosphor technology and the digitised images subjected to rigorous analysis using the QUEST II software system. Seventeen proteins whose relative synthesis decreased and four proteins that increased upon NGF withdrawal were located and are documented.
Two‐dimensional electrophoresis of thylakoid protein patterns in two wheat cultivars with different sensitivity to sulfur dioxide
Authors: Ranieri Annamaria , Castagna Antonella , Bini Luca , Soldatini Gian Franco 
Published: May 2005

AbstractPlants of two wheat cultivars (Triticum aestivum L., cultivars Mec and Chiarano), differing in sensitivity towards sulfur dioxide, were fumigated with 96 nL L−1 of SO2 for four months. Thylakoid membranes were subjected to two‐dimensional electrophoresis in order to detect possible changes in the protein patterns. We found the greatest alterations following treatment in the polypeptide profiles of the most sensitive cultivar, Mec, even if some significant changes were also detected in the plants of the cultivar Chiarano.
A comparison of resolution of DNA fragments between agarose gel and capillary zone electrophoresis in agarose solutions
Authors: Weiss George H. , Garner Mark , Yarmola Elena , Boček Petr , Chrambach Andreas 
Published: May 2005

AbstractThe resolving power of capillary zone electrophoresis (CZE) is compared to that of gel electrophoresis (GE) under similar conditions (agarose, similar length of DNA fragments, identical buffer) but with differences in temperature and field strength. The comparison is based on the time required to reach a desired degree of resolution by each of the two methods. A resolution parameter is developed which is equally applicable to CZE, with relatively diffuse initial conditions in the absence of stacking and measurements expressed in terms of time, and to GE, in which measurements are expressed in terms of spatial parameters. The resolution time in CZE using agarose solutions at 40°C was found to be greater by at least one order of magnitude than that in GE using agarose gels. Thus, the increased migration velocity due to high field strength in CZE substantially outweights the lower dispersion in GE.
Some reminiscences of the history of electrophoresis
Authors: Rilbe Harry 
Published: May 2005

AbstractDole's and Longsworth's solution of the whole set of moving boundary equations by using the dimensionless transference numbers is reviewed. Longsworth's teachings on the nature of transference numbers and his pondering over transference numbers of proteins led the author to inquire why transference numbers of ampholytes were never measured. Logical reasoning on this subject led to the invention of isolectric focusing.
From image processing to classification: III. Matching patterns by shifting and stretching
Authors: Skovgaard Ib Michael , Jensen Kirsten , Søndergaard Ib 
Published: May 2005

AbstractA method for the classification of electrophoretic patterns is described and tested on a data set representing ten wheat varieties. The method attempts to match each electropherogram to each variety by a transformation involving displacement and stretching along the x‐axis. This is done essentially by the method of least squares, which uses only the information contained in the electropherogram itself to adjust it to the variety in question. The method is completely automatic and works extremely well by classifying 98% of the spectra correctly, judged by cross validation.
Chemical stability of polyacrylamide‐coating on fused silica capillary
Authors: Nakatani Manabu , Shibukawa Akimasa , Nakagawa Terumichi 
Published: May 2005

AbstractThe chemical stability of polyacrylamide coatings on a fused silica capillary for capillary electrophoresis was investigated with respect to temperature of capillary and pH as well as salt concentration of running buffer solution by evaluating their effects on electroosmotic mobility. The capillary covalently bonded to linear polyacrylamide through Si‐C linkage was stable in buffer solutions of pH 2.3–8.0 at 30°C for 30 days, whereas the capillary bonded through Si‐O‐Si‐C linkage was damaged at pH above 4.6. The electroosmotic mobility observed in both capillaries increased with increasing temperature, though the degree of increase was smaller in the Si‐C linked capillary than in the Si‐O‐Si‐C linked capillary. The increase in the buffer concentration resulted in increased electroosmotic mobility in the Si‐O‐Si‐C linked capillary, but no effect was observed for the Si‐C linked capillary.
Effect of organic modifier concentrations on electrokinetic migrations in micellar electrokinetic chromatography
Authors: Chen Nong , Terabe Shigeru , Nakagawa Terumichi 
Published: May 2005

AbstractThe effect of low concentrations of organic modifiers on the electroosmotic mobility, μeo, and electrophoretic mobility of the micelle, μep, mc, in micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulfate (SDS) solutions was investigated. The results showed that μeo was mainly influenced by the viscosity of the running solution. A linear relationship between μeo and the concentration of the organic modifier was found in MEKC. A newly defined parameter, the mobility ratio, Rm, mc, which is the ratio of the electro‐phoretic mobility of the micelle to the electroosmotic mobility, and which characterizes the surface charge density of the micelle in MEKC system with organic modifiers, was introduced. A linear relationship between Rm, mc and the organic modifier concentration was observed. The parameter Rm, mc was found to be useful to investigate the changes in the micellar phase with the addition of organic modifier. The measured μep, mc values showed different and interesting characteristics among different organic modifiers.
Fluorescence imaging detection for capillary isoelectric focusing
Authors: Wu Xing‐Zheng , Wu Jiaqi , Pawliszyn Janusz 
Published: May 2005

AbstractA simple laser‐induced fluorescence (LIF) imaging detector and an ultrasensitive LIF imaging detector are described for capillary isoelectric focusing (CIEF). An argon ion laser beam of 496.5 nm is used as excitation source. In the simple LIF imaging detector, the excitation beam is directed into a capillary column by an optic fiber array. In the ultrasensitive LIF imaging detector, the laser beam is first expanded, then is focused into the 4.5 cm long capillary column by a cylindrical lens. Fluorescence emission is detected by a charge‐coupled device (CCD) camera. The feasibility and performance of the LIF imaging detector system for CIEF are first verified with a naturally fluorescent protein, b‐phycoerythrin. Then, the ultrasensitive LIF imaging system is used as a detector for CIEF of proteins labeled with fluorescein isothiocyanate (FITC). Three FITC‐labeled proteins (i) α‐D‐galatosylated FITC‐albumin, (ii) insulin‐FITC, and (iii) casein‐FITC, are used as model samples. Fluorescence images of the model samples are measured during the CIEF process. The focusing of the protein samples is complete in about 1.5 min. The ultrasensitive detector's detection limits for the FITC‐labeled proteins are at the level of 10−10 M, and the mass detection limits are about 4.5 × 10−17 mole, even though only 10% of the fluorescence emission is collected. Therefore, the method is capable of separating and detecting 10−11 M or amole (10−18 mole) level protein samples with a band‐pass filter more specific to the fluorescence light. Potential applications of the LIF imaging system in addition to quantitation of separated fluorescent species in various capillary electrophoresis methods can also include investigation of interaction between analytes focused in a capillary column.
Fluorescence‐labeled peptides as isoelectric point (pI) markers in capillary isoelectric focusing with fluorescence detection
Authors: Shimura Kiyohito , Kasai Ken‐Ichi 
Published: May 2005

AbstractCommercially available peptides, mostly angiotensin derivatives, were labeled at their N‐terminal amino group with 5‐carboxytetramethylrhodamine succinimidyl ester, to obtain fluorescent pI markers for capillary isoelectric focusing with fluorescence detection. The labeled peptides were purified by reversedphase chromatography. They were well separated on isoelectric focusing in a polyacrylamide gel slab and their pIs were determined by comigration with protein‐pI markers. The fluorescent markers could be detected as sharp peaks in capillary isoelectric focusing with laser‐induced fluorescence detection (He‐Ne laser, 1 mW, 543.5 nm). The detection limit was found to be around 3 × 10−12 M (0.8 amol). Tetramethylrhodamine‐labeled pea lectin (3 pg) was subjected to capillary isoelectric focusing and the pIs of the fluorescent derivatives of the lectin were determined by using the fluorescence‐labeled peptides as pI markers.