bluuebunny/crossref_metadata_2025_split huggingface dataset:
Highly cationic anti‐DNA antibodies in patients with lupus nephritis analyzed by two‐dimensional electrophoresis and immunoblotting
Authors: Kohro‐Kawata Junko , Wang Pei , Kawata Yasunobu , Matsuzaki Masunori , Nakamura Kazuyuki 
Published: May 2005

AbstractThe relationship between chemical properties of anti‐DNA antibodies (Abs) and lupus nephritis was investigated. The anti‐DNA Abs in sera from systemic lupus erythematosus (SLE) patients were separated by two‐dimensional electrophoresis (2‐DE) and immunoblotting with goat anti‐human IgG Abs. Highly cationic anti‐DNA Abs were detected in deoxyribonuclease I (DNase I)‐treated sera from patients with lupus nephritis (in 8 of 9 cases) but not in the sera from SLE patients without nephritis (in 0 of 9 cases), normal subjects, or patients with other renal diseases (in 0 of 7 cases). The mean titers of anti‐dsDNA Abs in patients with lupus nephritis were not significantly different from those in SLE patients without nephritis. The highly cationic anti‐DNA Abs in the sera disappeared after incubation with heparin‐Sepharose. These results suggest that highly cationic anti‐DNA Abs are specific for lupus nephritis and may be involved in development of lupus nephritis via the binding to glycosaminoglycans on the endothelial cell surface.
In memory of Harry Rilbe: An outstanding scientist who made many valuable contributions to the development of electrophoretic methods
Authors: Vesterberg Olof 
Published: May 2005

AbstractIn July 1997 Professor Harry Rilbe passed away at the age of 84. Harry Rilbe was an outstanding personality skilled in chemistry, optics and mathematics, and he will be remembered for his important contributions to the development of separation methods, notably moving boundary electrophoresis and isoelectric focusing. The latter method is used for analytical and preparative purposes in thousands of laboratories worldwide. Isoelectric focusing is indispensable for characterization of proteins from humans, animals, plants and microorganisms. Isoelectric focusing is thus useful for the understanding of the function of genes and, accordingly, for progress in the life sciences. A biographical retrospect, “A scientific life with chemistry, optics and mathematics”, was published by Harry Rilbe in Electrophoresis 1984, 5, 1–17.
Theory and practice of isoelectric focusing of interacting systems
Authors: Cann John R. 
Published: May 2005

AbstractThe theory of mass transport coupled to reversible protein interactions forms the basis for computer simulation of the isoelectric focusing behavior of several model systems. These include pH‐dependent conformational transition, carrier ampholyte‐induced interactions and protein‐ligand interactions. The computational results compare favorably with experimental observations. In addition, a method is formulated for an isoelectric focusing procedure which enables determination of intrinsic ligand‐binding constants for statistical binding of a charged ligand, binding to heterogeneous sites, and cooperative binding.
Electrophoresis of proteins across a transverse sodium dodecyl sulfate gradient
Authors: Gianazza Elisabetta , Vignati Mara , Santi Ombretta , Vecchio Giuseppe 
Published: May 2005

AbstractWe describe a new protocol for denaturant gradient gel electrophoresis, namely the migration of proteins across transverse sodium dodecyl sulfate (SDS)‐gradients. We show how such gradients may be reproducibly cast, and demonstrate their stability with time once an appropriate SDS reservoir is arranged at the cathode. SDS affects both size and surface charge of the molecules, and influences the secondary and tertiary structure to a variable extent, even in opposite directions. Hence, distinct, sometimes complex, denaturation patterns may be observed for different proteins.
Rat urinary proteins binding human IgE detected by chemiluminescence
Authors: Bayard Cecilia , Vesterberg Olof 
Published: May 2005

AbstractWork with laboratory animals involves the increased risk of developing an allergy. Certain proteins in male rat urine have been shown to be major allergens, i.e., Rat n 1.01 and Rat n 1.02. Rat urinary proteins separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) were electroblotted to polyvinylidene difluoride (PVDF) membranes. Immunoblot analysis of human IgE binding components in rat urine utilizing chemiluminescence and a newly developed luminometer is described. Luminometer scanning curves of IgE reactivity to the separated rat urinary proteins are demonstrated. Rat allergic individuals showed different immune responses to the separated proteins. Rat n 1.02, also known as the major protein α2u‐globulin, was not always the dominant allergen. Another protein in the albumin region, 60–67 kDa, was found to be an important allergen to some rat‐sensitive subjects. Reactivity in the skin prick test with purified Rat n 1.01 and Rat n 1.02 fractions were strong. However, in dot blot under nondenaturing conditions, only weak responses were obtained to the purified rat urinary proteins except for the albumin fraction. Chemiluminescence measurements in blotting membranes of patient IgE bound to different dilutions of certain rat urine proteins revealed good quantitative relationships. Three different chemiluminescence substrates were tested. Measurement of IgE bound to individual allergens as well as the abundance and relative importance of various allergens were studied.
Use of a Hepta‐tyr glycopeptide antibiotic as chiral selector in capillary electrophoresis
Authors: Fanali Salvatore , Aturki Zeineb , Desiderio Claudia , Boss Alessandra , Righetti Pier Giorgio 
Published: May 2005

AbstractA new glycopeptide antibiotic, MDL 63,246 (Hepta‐tyr), of the teicoplanin family, has been evaluated in capillary electrophoresis for the resolution of chiral compounds of pharmaceutical and environmental interest. Electrophoretic separations were carried out in a polyacrylamide‐coated capillary using the partial filling‐counter current mode with aqueous‐organic buffers in the pH range 4–6. Experimental parameters affecting resolution, such as antibiotic concentration, buffer pH, organic modifier type and capillary temperature, were studied. The Hepta‐tyr antibiotic exhibited a high enantiorecognition capability towards the studied compounds at very low concentrations (1–2 mg/mL). The optimum experimental conditions were achieved by using a buffer at pH 5 containing acetonitrile at 25°C.
Clinical analysis of human urinary proteins using high resolution electrophoretic methods
Authors: Marshall Thomas , Williams Katherine M. 
Published: May 2005

AbstractThe application of isoelectric focusing (IEF), sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), two‐dimensional electrophoresis (2‐DE) and capillary electrophoresis (CE) for high resolution electrophoretic analysis of human urinary proteins is reviewed. In each case, the information is tabulated chronologically with details of sample preparation, electrophoretic system, detection method and clinical application. The text includes an historical perspective of the use of each method for urinalysis and a detailed review of the application of the methods to the investigation of renal disease, renal transplantation, Bence Jones proteinuria (BJP), diabetes mellitus, cadmium toxicity, nephrolithiasis and cancers of the urogenital tract.
Proteomic analysis of enamel cells from developing rat teeth: Big returns from a small tissue
Authors: Hubbard Michael J. 
Published: May 2005

AbstractPoor understanding of the molecular links between disturbed calcium regulation in cells and disease remains a problem of considerable biomedical importance. Dental enamel cells might provide useful insights to this problem since they handle calcium in bulk without suffering its cytotoxic effects. Two practical challenges hindered investigation of calcium handling mechanisms in enamel cells ‐ paucity of molecular information and limited availability of sample from developing rat teeth, the experimental system of choice. This paper outlines the microscale proteomic approaches we applied to overcome these difficulties and reviews several major findings that ensued from initial characterization of the enamel cell proteome. Enamel cells are now established as a powerful model for fundamental calcium research and have provided outcomes of broad biological relevance, including the discovery of a new endoplasmic reticulum protein. Future proteomic approaches that might benefit understanding of function are discussed.
Separation of neutral compounds by capillary electrokinetic chromatography using polyethyleneimine as replaceable cationic pseudostationary phase
Authors: Maichel Beate , Potocek Blahoslav , Kenndler Ernst , Gas Bohuslav , Chiari Marcella 
Published: May 2005

AbstractPolyethyleneimine (PEI, molecular weight 6 × 105 − 1 × 106) is applied as a positively charged pseudostationary phase for electrokinetic chromatography (EKC) of uncharged mono‐ and oligophenols. EKC is carried out in PEI‐coated fused‐silica capillaries (with electroosmotic flow directed towards the anode) in 2‐(N‐morpholino)ethanesulfonic acid (MES) buffer (pH 7.0,20 mM) with PEI added to the solution in concentrations up to 0.70% w/v. The pseudo‐stationary phase leads to a retardation of the solutes mainly according to the number (and the position) of the OH‐groups of the separands, and is not influenced significantly by methyl groups. For 0.70% w/v PEI solution, for instance, the relative retention, ϱ, has values between 0.33 and 0.53. For the systems with the highest resolution of the separands (0.25–0.30% PEI) 190000 plates per meter are observed. The results indicate that the separation selectivity is mainly caused by ion‐dipole interactions between the OH‐groups of the solutes and the pseudostationary phase.
Use of scavenger beads to remove excess labeling reagents from capillary zone electrophoresis samples
Authors: Mort Andrew J. , Zhan Dongfeng , Rodriguez Veronica 
Published: May 2005

AbstractIn many cases, samples for capillary zone electrophoresis (CZE) are derivatized with a chromophore or fluorophore to enhance their detectability. To ensure efficient labeling, a large excess of labeling agent is often used, which leads to the presence of a large peak for unreacted reagent. Here we report that excess reagent can be reacted with “scavenger beads” carrying an appropriate functional group to remove it from the sample solution. We present examples of removal of aminonaphthalene mono‐, di‐, and trisulfonic acid from mixtures in which they were used to label mono‐ or oligosaccharides by reductive amination. Aldehyde‐containing scavenger beads were made by oxidizing Sephadex G‐50 beads with sodium periodate. These were added to the labeling reaction mixtures after the reductive amination of the sugars was complete. Almost complete elimination of the peak from the labeling agent could be achieved.
Capillary electrophoresis of arsenic compounds with indirect fluorescence detection
Authors: Huang Yu‐Min , Whang Chen‐Wen 
Published: May 2005

AbstractA capillary electrophoresis (CE)‐indirect fluorescence detection method for arsenic compounds is described. The five arsenic species, viz., arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA), dimethylarsinate (DMA) and phenylarsonate (PhA), were efficiently separated by CE in 8 min with an 1.5 mM fluorescein solution at pH 9.8. Fluorescein also functioned as a background fluorophore for the indirect detection of these nonfluorescent arsenic species. Linearity (r>0.996) of more than two orders of magnitude was generally obtained. The relative standard deviation (RSD) values were in the ranges 0.4–0.7% and 2.2–8.2% for migration times and peak areas, respectively. The concentration limits of detection (CLODs) for the arsenic compounds studied were between 0.04 and 0.16 μ/mL (as arsenic). The detection sensitivity was generally dependent upon the transfer ratio (TR, defined as the number of moles of fluorescein ions displaced by one mole of analyte ions) of each arsenic species. The applicability of the method for the analysis of ground water was examined.
Factors influencing the choice of buffer in background electrolytes for indirect detection of fast anions by capillary electrophoresis
Authors: Doble Philip , Macka Miroslav , Haddad Paul R. 
Published: May 2005

AbstractThe suitability of relatively slow (low absolute value of mobility) coanionic buffers in background electrolytes (BGEs) for indirect photometric detection of anions by capillary electrophoresis was investigated. As a model system, 2‐(cyclohexylamino)ethanesulfonic acid (CHES) was used to buffer the indirect detection electrolyte of sodium chromate. CHES (pKa 9.55) is a zwitterionic molecule carrying a net negative charge depending on the pH (effective charge −0.5 at pH = pKa). Within its useful pH buffering range CHES acted as a competing probe coanion. System peaks were induced which had deleterious effects on the detection sensitivity of slow to medium mobility anions. The mobility of the system peak was determined by the effective mobility of CHES, both of which increased with increasing pH. The peaks of analytes that migrated near or on the system peak were distorted and lost all quantitative properties. Analytes that migrated after the system peak either were not detected or reversed their responses. Analytes that migrated well before the system peak were unaffected. Consequently, the suitability of slow coanionic buffers is limited either to (i) fast anions or, (ii) a pH range much below the pKa, where the buffering capacity is not optimal.
Chemiluminescence detection in integrated post‐separation reactors for microchip‐based capillary electrophoresis and affinity electrophoresis
Authors: Mangru Shakuntala D. , Harrison D. Jed 
Published: May 2005

AbstractChemiluminescence (CL) detection based on the horseradish peroxidase (HRP) catalyzed reaction of luminol with peroxide was investigated as a post‐separation detection scheme for microchip‐based capillary electrophoresis. An integrated injector, separator and post‐separation reactor was fabricated on planar glass wafers. The fluorescein conjugate of HRP (HRP‐F1) was used as a sample for optimization of the CL detector response. In devices etched 10 μm deep, with an aluminum mirror integrated onto the backside of the detection zone to enhance collection efficiency, the detection limit, estimated at 3 standard deviations (SD) above background noise, for 1 nL injected sample plugs was 35 nM in HRP‐F1. In devices etched 40 μm deep, 8 nL plugs gave a detection limit of 7 nM. Separation and CL detection of the products of an immunological reaction of a F(ab')2 fragment of the HRP conjugate of goat anti‐mouse immunoglobulin G (IgG) with mouse IgG was performed on‐chip. A linear calibration curve was obtained for the decrease in peak height of the HRP conjugate (53 μg/mL) with increasing mouse IgG (0–60 μg/mL). When microperoxidase was used as an internal standard, the R2 value of a linear least‐squares fit was 0.9867, and the relative errors in the slope and intercept were ± 5.8 and ± 1.3%, respectively.
Detection of isozymes of deoxyribonucleases I and II on electrophoresed gels with picogram sensitivity using SYBR green I
Authors: Iida Reiko , Tsubota Etsuko , Sawazaki Kazumi , Aoyama Masahiro , Matsuki Takasumi , Yasuda Toshihiro , Nakashima Yoshimitsu , Kishi Koichiro 
Published: May 2005

AbstractA highly sensitive method for detecting deoxyribonucleases (DNases) I and II on an electrophoresed gel is described. A dried agarose film sheet containing DNA as a substrate and a buffer reagent was placed in contact with the gel surface after electrophoresis (DAFO method, Yasuda et al., Anal. Biochem. 1989, 183, 84–88). After an appropriate incubation period, the film sheet was peeled off and stained with SYBR‐Green I (SG), and then the DNase isozyme bands were detected using a fluorescence image analyzer. We could detect pg levels of the DNases (DNase I, 2 pg; DNase II, 2pg), which represents a 32‐ to 128‐fold increase in sensitivity compared with the original DAFO method using ethidium bromide (EB) as the fluorescent dye. A combination of this new detection method and isoelectric focusing electrophoresis in polyacrylamide gel allowed accurate DNase I typing from 1 μL human serum. This new technique has been named SG‐DAFO, after its original dried agarose film overlay method using EB (EB‐DAFO).
Humic acid capillary zone electrophoresis adsorption on capillary walls, separation in metal ion supplemented buffer and the fingerprints
Authors: Fetsch David , Fetsch Martin , Mendéz Eladia M. Peña , Havel Josef 
Published: May 2005

AbstractCapillary zone electrophoresis (CZE) in quartz tubes is often being used for the separation and characterization of humic acids (HA). A method was found to follow adsorption (and kinetics) of humic acids on a fused‐silica capillary wall. It was shown that the adsorption of humic acids on an uncoated capillary wall is high. The effect on sorption of additives to the background electrolyte (BGE) was studied. Sorption can be eliminated by adding magnesium(II) salts (14–50 mM) to the BGE (pH 3.40) with resultant highly reproducible electropherograms as well as detailed and expressive fingerprints for HA of different origin.
Analysis of heat shock‐induced monophosphorylation of stathmin in human T lymphoblastic cell line JURKAT by two‐dimensional gel electrophoresis
Authors: Fujimoto Masanori , Nagasaka Yuji , Tanaka Tatehiko , Nakamura Kazuyuki 
Published: May 2005

AbstractTwo‐dimensional gel electrophoresis (2‐DE) was used to study alterations in intracellular proteins of the human T lymphoblastic cell line JURKAT by heat shock at 45°C for 30 min. The 2‐DE patterns indicated an increase in the amount of a spot of molecular weight (Mr) 18500 and isoelectric point (pI) 5.6, which was a monophosphorylated form of stathmin. Stathmin is a major substrate for a proline‐rich peptide‐specific serine protein kinase, a mitogen‐activated protein kinase, however, the enzyme was not activated by the heat shock. Further examinations of the effects of cAMP, phorbol myristate acetate, cyclosporin A, and staurosporine on phosphorylation suggest that cyclin‐dependent kinases might be responsible for the heat shock‐induced monophosphorylation of stathmin.
A survey of methodological challenges for glycosaminoglycan/proteoglycan analysis and structural characterization by capillary electrophoresis
Authors: Karamanos Nikos K. , Hjerpe Anders 
Published: May 2005

AbstractProteoglycans participate and regulate several physiological processes via their glycosaminoglycan constituents. For a deeper understanding of how they interact with extracellular ligands as well as with cell bound effector molecules, the fine chemical structures of their glycosaminoglycan chains must be elucidated. Lately developed capillary electrophoretic techniques is a powerful analytical tool for the analysis of glycosaminoglycans, combining a high resolving power with sensitive detection. In this review we describe how depolymerized and intact glycosaminoglycans/proteoglycans can be characterized by capillary electrophoresis, relating these analyses to their possible biological significance. Conditions for running these separations and the detection systems for particular applications are also summarized.
Investigation of micelles and anionic cyclodextrins as pseudostationary phases for the capillary electrophoresis separation of oligosaccharides derivatized with 2‐aminobenzamide
Authors: Tran Nguyet Thuy , Taverna Myriam , Deschamps Frantz S. , Morin Philippe , Ferrier Danielle 
Published: May 2005

AbstractOligomers of glucose and oligosaccharides released from glycoproteins were derivatized with 2‐aminobenzamide. As this fluorophore imparts no charge to the oligosaccharides, several strategies were investigated to achieve capillary electrophoresis (CE) separation of both neutral and charged derivatized glycans. Micellar electrokinetic capillary chromatography (MEKC) with the addition of anionic surfactants was evaluated as a first approach: sodium dodecyl sulfate (SDS) produced the best separation of the oligoglucose fragments, where the migration was inversely related to their degree of polymerization. To demonstrate the applicability of this method for complex carbohydrate analysis, oligosaccharide mixtures derived from ribonuclease B (RNase B) and α‐acid glycoprotein (α‐AGP) were analyzed. A satisfactory separation for the high‐mannose structures found in RNase B could be obtained, whereas charged oligosaccharides from α‐AGP were poorly resolved. Cyclodextrin‐modified CE was chosen as the second approach: the effect of the addition of sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) or sulfobutylether‐γ‐cyclodextrin (SBE‐γ‐CD) on the electrophoretic mobilities and resolution of neutral and charged oligosaccharides was then studied. Selectivity of sialylated structures could be further improved by using anionic cyclodextrins (CDs) instead of micelles. However, this latter approach failed to baseline‐resolve the different high‐mannose structures of RNase B. A successful separation of the complex mixture of oligosaccharides from α‐AGP was obtained with the addition of 4% of SBE‐γ‐CD and triethylamine (TEA) in a phosphate buffer, pH 6.7.
Isolation and characterization of β‐cyclodextrin sulfates by preparative gradient polyacrylamide gel electrophoresis, capillary electrophoresis and electrospray ionization—mass spectrometry
Authors: Hileman Ronald E. , Siegel Marshall M. , Tabei Keiko , Balagurunathan Kuberan , Linhardt Robert J. 
Published: May 2005

AbstractA β‐cyclodextrin sulfate mixture has been fractionated using discontinuous gradient polyacrylamide gel electrophoresis. Semidry electrotransfer of the sample onto a positively charged nylon membrane and visualization of a portion of this membrane with Alcian blue stain showed multiple bands. The bands were cut from the remaining portion of the membrane and after washing with 8 M urea, the β‐cyclodextrin sulfate fractions were eluted with 2 M sodium chloride and dialyzed. Analysis of each fraction using high resolution analytical gradient polyacrylamide gel electrophoresis as well as capillary electrophoresis, using indirect detection, showed some of the fractions to be pure while others were mixtures. Each β‐cyclodextrin sulfate fraction was complexed with a basic synthetic peptide and analyzed by electrospray ionization mass spectrometry to define the mass of the components in each mixture and thereby to determine the purity of each sample.
Interval isotachophoresis for purification and isolation of ionogenic species
Authors: Weber Gerhard , Boček Petr 
Published: May 2005

AbstractA new preparative electrophoretic method in free solution is described, consisting of three consecutive steps: (i) filling the separation chamber with a suitable electrolyte system and sample in parallel streams by laminar hydrodynamic flow, (ii) applying the voltage across the chamber with isotachophoretic separation for a definite time interval operating in the direction perpendicular to that of filling, (iii) reapplying a hydrodynamic flow (without voltage) and collecting the separated species via an array of outlets. This approach completely eliminates the main drawback of the continuous flow electrophoresis (CFE), i.e., the electrohydrodynamic distortion of zones. This method utilizes the instrumentation devised for CFE and enables the isolation of large amounts of individual sample species comparable to that of CFE, with a resolution comparable to that of capillary isotachophoresis. The precise timing of the consecutive steps in the procedure as well as the stability of the operational parameters are of key importance for reproducibility. By using cationic isotachophoresis with 3 synthetic pI markers as model sample species, the reproducibility, stability and the separation power of the newly presented method are demonstrated. The sample throughput corresponds to micromoles per hour.