bluuebunny/crossref_metadata_2025_split huggingface dataset:
The potentiometric stripping analysis of proteins
Authors: Honeychurch Michael J. , Ridd Michael J. 
Published: May 2005

AbstractTen proteins were analyzed by potentiometric stripping analysis (PSA) following reductive accumulation on a HMDE and oxidation with dissolved oxygen. During the reductive accumulation period the surface disulfide bonds are reduced to sulfhydryl pairs. The rate of oxidation of the sulfhydryl pairs is determined by the transport of dissolved oxygen to the electrode surface. Comparison of the PSA response to that obtained by constant current chronopotentiometry (CCCP) of the reduced protein in a deoxygenated solution showed that the presence of dissolved oxygen, in the PSA experiments stabilized the proteins from further conformational changes following the reduction of the surface disulfide bonds.
Investigation into electrochemical pretreatment of carbon fiber electrodes for the analysis of bamipine and imipramine
Authors: Surmann Peter , Peter Bernhard 
Published: May 2005

AbstractThe influence of the parameters of electrochemical pretreatment of carbon fiber electrodes on the differential pulse voltammetric response of bamipine and imipramine is described. Upon increasing the pretreatment potential a slight activation is followed by a deep suppression of peak current in the analysis of both substances. After a turning point the currents rise again and exceed the initial values many times over. The peak potential is affected as well. A slight deactivation is followed by a greater activation. The surfaces were characterized by chronoamperometry, chronopotentiometry and inverse voltammetry of the electrode material. At the carbon fiber epoxy matrix electrode it is possible to distinguish three different stages, which are interpreted as a phase of slightly activating functional groups in a submonolayer, a phase of passivating multilayer oxidation and finally a strongly activating phase of burn‐off and consumption of the electrode surface with change in its structure. These are thought to be responsible for the effects at the surface. It was shown that the effects within the third phase are diametrically opposed for aromatic and aliphatic amines.
Photo‐electroanalysis: Indirect determination of anthracene using the oxygen reduction in aqueous acetonitrile
Authors: Eklund John C. , Hallick Alan , Kumbhat Sunita , Compton Richard G. 
Published: May 2005

AbstractA novel method for the photoelectroanalysis of anthracene, as a model compound, is proposed. This technique utilizes a gold electrode in a flowing system of electrolyte, air saturated with oxygen. On irradiation of the electrode with UV‐vis light there is a reduction in the level of the oxygen four‐electron reductive limiting current due to a reaction of the excited state anthracene and oxygen in acetonitrile/water solution. A linear response over four orders of magnitude (10−2 to 10−1 mol dm−3 was observed between this reduction of current and the concentration of anthracene suggesting the application of this type of technique to the detection of trace levels of aromatic molecules.
Analysis of skin fibroblast proteins in Duchenne muscular dystrophy: 1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Authors: Burghes Arthur H. M. , Dunn Michael J. , Statham Helen E. , Dubowitz Victor 
Published: May 2005

AbstractProtein profiles of skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) and normal individuals have been compared using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) combined with various detection systems. Protein patterns of normal and DMD fibroblasts obtained usin 10 % polyacrylamide gels showed no qualitative or quantitative differences. The use of acrylamide gels results in increased resolution. No qualitative or quantitative differences were observed using Coomassie Brilliant Blue R‐250 staining. However, Coomassie Brilliant Blue lacks the sensitivity to detect many minor components. Therefore a silver staining procedure was used, which resulted in greatly increased detection sensitivity. Background staining did not increase with gel concentration, but stain development proceeded more slowly at higher acrylamide concentrations. No qualitative differences were observed between normal and DMD profiles. Fibroblasts radiolabelled with [35S]‐methionine were analysed using gradient gels. No qualitative differences between DMD and normal patterns were observed by autoradiography of dried gels. However, quantitative analysis of slicing of gel rods revealed differences between normal and DMD fibroblasts. Two methods of statistics were used to analyse this data, firstly a method that accounts for inter‐experimental variation (termed “batch statistics”) and secondly the standard t‐test. Regions of 73 000 to 68 000 and 48 000 molecular weight were decreased in DMD samples and this finding was significant by both methods of statistical analysis. Regions of 175 000 and 53 000 molecular weight were significantly elevated in DMD preparations only by t‐test, whereas a regions of 32 × 103 molecular weight was only significantly elevated when tested by batch statistics.
Analysis of skin fibroblast proteins in Duchenne muscular dystrophy: 2. Isoelectric focusing under dissociating conditions
Authors: Burghes Arthur H. M. , Dunn Michael J. , Statham Helen E. , Dubowitz Victor 
Published: May 2005

AbstractProtein profiles of skin fibroblasts labelled with [35S]‐methionine from patients with Duchenne muscular dystrophy (DMD) have been compared with those of cells from normal individuals by isoelectric focusing on thin polyacrylamide gels containing denaturing and solubilising agents cast on silanized supports. The routine method of sample solubilisation used a mixture of 8 M urea and 2 % NP‐40. The inclusion of sodium dodecyl sulphate (SDS) in the sample was found to result in removal of non‐ionic detergent from the gel even if a competition step had been used. In the presence of SDS less material remained at the point of sample application with a concomitant increase in bands in the middle region of the gels. Gels containing a mixture of sulfobetaine and NP‐40 were found to be inferior to those with 8 M urea and 2 % NP‐40, and high levels of urea were found to precipitate the zwitterionic detergent. No consistent qualitative differences between normal and DMD patterns were observed using any of the solubilisation methods on pH 3 to 10 or pH 8 to 10.5 gels. In order to facilitate quantitative analysis by gel slicing techniques, a plastic sheet was developed which would reliably bind acrylamide in the presence of 8 M urea and 2 % NP‐40. The quantitative analysis revealed significant differences between normal and DMD profiles, but the relevance of these changes to the disease state awaits further investigation.
Quantitative aspects of silver deposition in proteins resolved in complex polyacrylamide gels
Authors: Guevara Juan , Johnston Dennis A. , Ramagali Louis S. , Martin Barbara A. , Capetillo Sylvia , Rodriguez Lewis V. 
Published: May 2005

AbstractDeposition of silver or silver compounds in proteins separated in linear gradient polyacrylamide electrophoretic gels is influenced by protein concentration and location, polyacrylamide concentration, extent of washing after incubation in ammoniacal silver hydroxide solution, and development time in a citric acid: formaldehyde solution. Our modification of existing silver staining methods provided routine resolution of polypeptides separated in 1.0 to 1.5‐mm‐thick gels without high background staining or surface staining and with picogram detection sensitivity. Removing electrophoretic components, such as sodium dodecyl sulfate (SDS), sulfhydryl reagents (2‐mercaptoethanol, dithiothreitol and dithioerythreitol), glycerol, tris‐glycine, carrier ampholytes, urea, and acetic acid, was essential in order to attain silver staining of proteins with a negligible background. Ammoniacal silver ions or silver ions, associated and unassociated with proteins separated in polyacrylamide gels, were removed during the washing step following incubation in ammoniacal silver hydroxide solution. Loss of stain intensity at discrete protein clusters was shown to correlate with polyacrylamide concentration in gradient gels. Standardizing the washing step enhanced reproducibility of staining. Using this silver staining method and computer‐assisted image processing, it was possible to detect quantitatively as little as 27 picograms of protein per mm in 10 %‐20 % linear gradient polyacrylamide gels (1.2 mm thick). Based on the lowest detection limits for Coomassie Brilliant Blue R‐250 staining of protein (10 nanograms), this method for silver staining is at least 370‐fold more sensitive.
Silver stain after isoelectric focusing of unconcentrated cerebrospinal fluid: Visualization of total protein and direct immunofixation of immunoglobulin G
Authors: Confavreux Christian , Gianazza Elisabetta , Chazot Guy , Lasne Yves , Arnaud Philippe 
Published: May 2005

AbstractA silver stain technique has been developed to study proteins in unconcentrated cerebrospinal fluid (CSF) after isoelectric focusing. This method is highly sensitive, and bands containing 25 to 50 ng protein can be clearly distinguished, so that small volumes (40 μl maximum) of native CSF can be used. Individual proteins (e. g., immunoglobulin G) can be detected easily by specific immunofixation. It is also possible to perform direct precipitation and direct specific immunofixation on a single gel in order to compare side by side the patterns of the whole and of specific proteins from different samples of CSF. The technique is simple and highly reproducible, and results are obtained 6 h after sample deposition (if immunofixation is used, a further 24 h washing is necessary). The sensitivity and versatility of this technique (immunofixation can be applied to the detection of any antigen) should permit its extension to other biological fluids with a low protein content.
A fast flat‐bed laser scanner for the digital acquisition of two‐dimensional electrophoresis patterns
Authors: Marsman Herman , van Resandt Roelof Wijnaendts 
Published: May 2005

AbstractAnalysis of two‐dimensional electrophoresis patterns demands computer assistance. Specialized machines to read and store the pattern of an autoradiograph or of a “wetgel” into a digital memory are rare. A fast and flexible opto‐mechanical picture reading and storage machine, specially designed for the computer‐aided analysis of two‐dimensional electrophoresis patterns, has been constructed. It is now possible to digitize, for example, a picture of 250 × 400 mm into pixels of ca. 250 × 250 μm with a grey level resolution of 8‐bit within 70 s. Digitizing at significant pixel sizes down to 64 μm is possible at correspondingly longer acquisition times for the areas to be digitized. The design is a flat‐bed scanner. One axis is scanned optomechanically (Galvanometer scanned laser beam), the other axis is scanned mechanically. The area covered by the scanning laser beam has a size of 250 × 400 mm or smaller. The control of the scanner, including pixel density and sampling synchronization is executed by a microprocessor system.
Design of a vertical gel slab electrophoresis apparatus containing a permanent salt bridge
Authors: Schafer‐Nielsen Claus 
Published: May 2005

AbstractAn electrophoresis apparatus of the vertical slab gel type was designed to obtain a unit in which the number of spare parts and manual operations necessary for its assembly is significantly reduced as compared to the designs that are commonly used at present. The new apparatus is assembled in less than 60 s, and the gels are moulded in situ., i. e. without the use of a separate casting stand. With all electrode reservoirs placed within the base unit, the apparatus obviates the need for bulky external electrode reservoirs. The compact design is made possible by use of a permanently mounted porous plastic bridge that acts as a seal during moulding of the gel and as a conducting salt bridge during electrophoresis. The apparatus permits simultaneous running of two gels, and runs on single gels may be performed independently without filling the electrode reservoirs for the other gel.
An evaluation of DNA fingerprinting for forensic purposes
Authors: Gill Peter , Lygo Joan E. , Fowler Susan J. , Werrett David J. 
Published: May 2005

AbstractMany highly polymorphic minisatellite loci can be detected simultaneously in the human genome by hybridisation to probes consisting of tandem repeats of the ‘core’ sequence. The resulting DNA fingerprints produced by Southern blot hybridisation are comprised of multiple hypervariable DNA fragments, show somatic and germline stability and are completely specific to an individual. DNA of high molecular weight can be isolated from blood and semen stains up to 4 years old. Sperm nuclei can be separated from vaginal debris and the sperm DNA examined in isolation, allowing the positive identification of rapists. Correspondingly, vaginal DNA can be isolated from extracts of penile swabs. DNA can also be isolated from extracts of buccal swabs. A blind trial involving 43 blood samples, 11 bloodstains and 11 semen stains has been successfully carried out. It is envisaged that genetic fingerprinting will shortly form an important part of casework procedure in forensic science laboratories.
Effect of ligand‐affinity differences of human hemoglobin variants on electrophoretic behavior and their isolation and functional characterization
Authors: Rochette Jacques , Deburgrave Nathalie , Bohn Brigitte , Dodé Catherine , Poyart Claude , Krishnamoorthy Rajagopal 
Published: May 2005

AbstractA natural sulfated polysaccharide (agaropectin), contained in crude agar, can be used as a medium for electrophoretic separation of hemoglobin mutants, constituting a particular class of protein‐ligand interactions. Mutations which either modify the electrostatic charge at the surface of the hemoglobin molecule or not, have been studied according to their putative interaction with the medium. Using conformational specificities of the hemoglobin molecule, we have also demonstrated that isoelectric focusing on a polyacrylamide gel in the absence of heme ligands represents a useful, convenient and rapid procedure for isolating silent Hb variants in their native form, provided that they exibit an abnormal Bohr effect.
Characterization of human catalase by isoelectric focusing in presence of urea
Authors: Alonso Antonio , Visedo Guillemo , Sancho Manuel , Fernández‐Piqueras José 
Published: May 2005

AbstractHuman catalase from erythrocytes and liver were analyzed by polyacrylamide gel isoelectric focusing in presence and absence of urea using two different pH gradients, namely pH 6–8 and pH 6.7–7.7. In presence of urea, human catalase focused in the pH range 6.75–7.0, slightly anodal to that of hemoglobin A. In narrow pH gradients, human erythrocyte catalase was microheterogeneous. Neuraminidase from different sources and peptide‐N‐glycosidase F were applied to investigate the presence of sialic acid and/or carbohydrate chains in human catalase. A shift in the focusing pattern of both erythrocyte and liver catalase towards the anode was observed after treatment with one of the commercially available neuraminidase preparations. This unusual result could be related to a contaminating protease since no effect was observed when the catalases were treated in presence of a serine protease inhibitor. In contrast, bovine liver and Maccaca erythrocyte catalase did not display any detectable change in their focusing patterns after treatment with any of the neuraminidase preparations.
Two‐dimensional electrophoresis of membrane proteins: Separation of myelin proteins
Authors: Person Håkan , Överholm Torsten 
Published: May 2005

AbstractA method for two‐dimensional electrophoretic separation of myelin proteins is presented. The first dimension consists of isoelectric focusing of lyophilized and delipidated membrane proteins, solubilized in a mixture of the nonionic detergent Triton X‐100, the zwitterionic detergent CHAPS, 9 M urea and carrier ampholytes, and incorporated into a slab before separation. Subsequent discontinouous sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was performed ny moulding the isoelectric focusing slab gel with its supporting glass plate into the stacking gel. This method proved to give highly reproducible results since mechanical forces and thus the risk of stretching, folding or rupture of the isoelectric focusing slab gel is minimized. Furthermore, by immunoblotting. the positions of myelin‐associated glycoprotein and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase were established with specific antibodies.
High‐performance human myocardial two‐dimensional electrophoresis database: Edition 1996
Authors: Müller Eva‐Christina , Thiede Bernd , Zimny‐Arndt Ursula , Scheler Christian , Prehm Joachim , Müller‐Werdan Ursula , Wittmann‐Liebold Brigitte , Otto Albrecht , Jungblut Peter 
Published: May 2005

AbstractThe master gel of the human myocardial two‐dimensional electrophoresis (2‐DE) gel database contains about 3300 protein spots characterized in terms of isoelectric point (pI) and molecular mass. A high‐performance technique was applied, using large gels (23 × 30 cm). Isoelectric focusing with anodic sample preparation and nonequilibrium running conditions (NEPHGE) was combined with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in 15% acrylamide gels in the second dimension. The range of pI extends from pH 4.5 to 9.6. Seventy proteins were identified by combinations of amino acid analysis, N‐terminal and internal sequencing, immunostaining, matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) peptide mass fingerprinting, post‐source decay MALDI‐MS and ladder sequencing by carboxypeptidase P. The identification of additional proteins, not found in the master gel, was achieved by immunoblotting. Unequivocal identification with high sensitivity and good yield was obtained by combining internal sequencing and MALDI‐MS. In‐gel digestion, the concentration and purification of peptides in a peptide collecting device, and the improved FRAGMOD program for peptide mass fingerprinting have added to the security and sensitivity of identification. The high‐performance human myocardial 2‐DE database was built up with proteins detected by the TOPSPOT program. Spots within six sections of the whole pattern are clickable. Protein description includes detailed information about identification, characterization, and links to the related SWISS‐PROT, other 2‐DE databases and Medline entries. The database is constructed in accordance with four of the rules for a federated database.
Endosomal fractions from viral K‐ras‐transformed MDCK cells reveal transformation specific changes on two‐dimensional gel maps
Authors: Huber Lukas A. , Pasquali Christian , Gagescu Raluca , Zuk Anna , Gruenberg Jean , Matlin Karl S. 
Published: May 2005

AbstractWe have investigated the effects of viral Kirstenrasoncogene expression in Madin‐Darby canine kidney (MDCK) II epithelial cell on the differential protein expression of organelle proteins. MDCK cells, stably transformedviainfection with a helper‐independent retroviral vector construct, were grown on permeable filter supports. Whereas normal cells form highly polarized monolayers,ras‐transformed cells display an unpolarized phenotype, detaching from the substratum and developing multilayers (Schoenenberger, C.‐A.et al.,J. Cell Biol.1991,112, 873–889). We postulate that this breakdown of epithelial polarity reflects disturbed intracellular protein transport and sorting, namely, proteins will no longer be sorted correctly in intracellular organelles and will therefore not reach their appropriate target membrane. Here we emphasize the role of endosomes as sorting platform in epithelial cells. We found significant differences in the molecular composition of endosomes from normalvs.oncogenic transformed epithelial cells, strengthening previous evidence indicating that oncogenic transformation results in abnormal expression of normal genes (Celis, J. E., Olsen, E.,Electrophoresis1994,15, 309–344) as well as the expression of new ones (Huber, L. A.et al.,Electrophoresis1994,15, 468–473).
Use of capillary electrophoresis methods to characterize the pharmacokinetics of antisense drugs
Authors: Chen Shu‐Hui , Gallo James M. 
Published: May 2005

AbstractAs antisense drugs become mature for clinical trials, analytical techniques to analyze antisense DNA in biological media for characterization of their pharmacokinetics will be in demand. Due to the superior resolving power of capillary gel electrophoresis (CGE), CGE will likely be a preferred method in quantifying intact oligonucleotides as well as the putative metabolic products. Nonetheless, biological mediums can influence the stability of the gel column, making a CGE assay time‐consuming. In one approach, high‐performance liquid chromatography (HPLC) was used to quantify the total amount of antisense compounds to increase the sample throughput and CGE was used to determine the relative percentage of the intact and metabolic species on specific samples. Alternatively, extensive sample pretreatment procedures were performed and the samples were quantified and characterized directly by CGE alone with the use of an internal standard. Both methods have been used to characterize the pharmacokinetics of antisense compounds. This review focuses on the instrumental and technical aspects of analyzing antisense DNA in biological mediums using CGE either as a single or a combined method towards better understanding of the pharmacokinetics of antisense DNA. Moreover, the newer analytical technologies of capillary electrophoresis (CE), which hold great potential to be used for pharmacokinetic applications, such as the replenishable sieving matrix combined with an innovative coupling approach and microchip CE, will also be explored.
Analysis of cefadroxil by micellar electrokinetic capillary chromatography: Development and validation
Authors: Li Yong‐Min , Vanderghinste Dominique , Pecanac Darka , Schepdael Ann Van , Roets Eugène , Hoogmartens Jos 
Published: May 2005

AbstractA method is developed and validated for analysis of the antibiotic cefadroxil using micellar electrokinetic capillary chromatography. It permits cefadroxil to be completely separated from ten of its known related substances within 15 min (including the washing procedure). The separation is performed in an acetate buffer (50 mM, pH 5.25) containing sodium dodecyl sulfate (SDS; 110 mM). The fused‐silica capillary was 44 cm long (36 cm effective length), 50 μm ID; the voltage, 18 kV; temperature, 15°C; and the detection wavelength; 254 nm. The influence of the type of buffer, buffer pH and concentration, and of the SDS concentration was investigated. The robustness of the method was examined by means of a full‐fraction factorial design. The parameters for validation such as linearity, precision, limit of detection and limit of quantitation are also reported.