bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of heparin‐binding to lactoferrin from human milk and from human granulocytes by means of affinity capillary electrophoresis
Authors: Heegaard Niels H. H. , Brimnes Jens 
Published: May 2005

AbstractHuman lactoferrin from two different sources: milk and secondary granules of neutrophil granulocytes, was compared with respect to heparin binding by means of affinity capillary electrophoresis. This method is based on analysis of migration pattern shifts induced by various amounts of ligand present in the electrophoresis buffer. Since lactoferrin is a basic molecule, analyses were performed at pH 8 in a capillary with a neutral hydrophilic coating. However, analyzable peaks were only obtained in the presence of heparin in the electrophoresis buffer. Proportionally to the amount of heparin present, these peaks exhibited shape changes and strong migration shifts. In the ensuing analysis it could be demonstrated that the two lactoferrins had comparable migration shifts and peak shape changes. This indicates that also in this respect the two forms of lactoferrin are identical. Affinity capillary electrophoresis is a convenient and fast method to investigate functional characteristics of low amounts of unmodified biomolecules.
Optimization of selectivity and resolution in micellar electrokinetic capillary chromatography with a mixed micellar system of sodium dodecyl sulfate and sodium cholate
Authors: Wiedmer Susanne K. , Jumppanen Juho H. , Haario Heikki , Riekkola Marja‐Liisa 
Published: May 2005

AbstractSelectivity and resolution were studied for the separation of seven corticosteroids by micellar electrokinetic capillary chromatography (MEKC) using a mixed micellar solution of sodium dodecyl sulfate (SDS) and sodium cholate (SC), buffered with 3‐(N‐morpholino)propanesulfonic acid (MOPS) or 3–[(1,1‐dimethyl‐2‐hydroxyethyl)amino]‐2‐hydroxyoropane sulfonic acid (AMPSO). The changes in selectivity were compared for the AMPSO‐SDS‐SC system by varying the pH and the concentrations of AMPSO, SDS and SC. The experimental design started with the central composite design and continued in a sequential manner. The optimum selectivity for the separation of the corticosteroids was calculated from the analyte migration times and the analyte velocities, by using empirical quadratic regression models. Satisfactory regression fits and coefficients of determination for prediction were obtained with crossvalidated models. To optimize the resolution, the physical parameters of capillary length and analysis time were varied under the conditions optimal for the selectivity. In both the selectivity and the resolution, optimization the overall optimum was determined by using the desirability function technique. Analysis times were controlled by using 1,3‐diaminopropane to influence the electroosmotic flow velocity (Veo). The voltage was kept constant, which resulted in higher electric field strength in shorter capillaries. No changes in the selectivity were observed when 1,3‐diaminopropane was used to control the electroosmotic flow velocity. Such an optimization technique, where the chemical and physical factors affecting the separation are treated independently, seemed to be effective for finding the best possible resolution for the corticosteroids.
Sequencing in nanofabricated arrays: A feasibility study
Authors: Duke Thomas , Monnelly Glen , Austin Robert H. , Cox Edward C. 
Published: May 2005

AbstractThe feasibility of using nanofabricated arrays as electrophoretic chambers for DNA sequencing is investigated. A specific array design, consisting of rows of closely spaced posts, separated by longer open spaces, is proposed. Molecules driven through the array by an electric field get hooked over obstacles at successive rows and their progress through the device is delayed as a consequence. The dependence of the delay time on molecular size is derived. Numerical evaluation indicates that a device of modest dimensions, operating at high fields, can rapidly resolve oligonucleotides containing several hundred bases.
A rapid electrophoretic method for separating rabbit skeletal muscle myosin heavy chains at high resolution
Authors: Kubis Hans‐Peter , Gros Gerolf 
Published: May 2005

AbstractAn electrophoretic method using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was developed which gives a high‐resolution separation of the known myosin heavy chains of rabbit skeletal muscle with excellent reproducibility. The gel of 10 cm total length consists of (i) a first stacking gel of 3.5% total gel concentration (T) and pH 6.8, (ii) a first separating gel of 6.6%T and pH 8.8, (iii) a second stacking gel of 6.6%T and pH 6.8, and (iv) a second separating gel of 8.8%T and pH 8.8. With this composition, a minigel system allows separation of six myosin heavy‐chain (MHC) isoforms at room temperature without cooling and within 8h. In agreement with previous reports, the isoforms appear in the sequence MHCemb, MHC IIa, MHC IId, MHCneo, MHC IIb, MHC I. A special advantage is the detectability not only of the adult but also of the embryonic and neonatal isoforms MHCemb and MHCneo.
Quantitative densitometry of proteins stained with Coomassie Blue using a Hewlett Packard scanjet scanner and Scanplot software
Authors: Vincent Sandra G. , Cunningham Peter R. , Stephens Newman L. , Halayko Andrew J. , Fisher John T. 
Published: May 2005

AbstractIn the present study we evaluated the performance of a software/scanner system that employed the Hewlett Packard (HP) ScanJet Plus and Scanplot Software for densitometric quantification of protein loads stained with Coomassie Brilliant Blue following sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Gels with bovine serum albumin (BSA) standards, ranging from 0.125 to 10 μg, were scanned using reflectance densitometry with 127 μm step size in both the x and y directions and a resolution of 200 dots per inch. Densitometric volume was calculated for each protein band from scanner output in the tagged image file format (TIFF) by a customized software package, Scanplot V. 4.05 (Cunningham Engineering). Protein loads between 0.125 and 10.0 μg vs. volume were fit by a second‐order regression: Volume = −0.58 × protein load2 + 16.82 × protein load + 7.87 (r = 0.991, p < 0.01). The same gels were scanned and quantified using a transmittance laser densitometer; densitometric volumes measured by both systems were highly correlated (r2 = 0.981, p < 0.01). Additional gels of BSA, smooth muscle myosin heavy chain (myosin), and actin displayed linear relationships between protein loads up to 4.0 μg and densitometric volume reflecting unique dye binding properties. We conclude that accurate and reproducible quantitative densitometry of SDS‐PAGE can be performed using the HP ScanJet Plus scanner and Scanplot software.
Deoxyribonuclease I‐facilitated electrotransfer of protein‐DNA complexes from electrophoretic gels to nitrocellulose membranes
Authors: Ivanchenko Maria , Zlatanova Jordanka 
Published: May 2005

AbstractA simple and reproducible technique for efficient transfer of protein‐DNA complexes from electrophoretic gels to nitrocellulose membranes is described. Transfer of DNA‐protein complexes may be difficult, especially when the DNA is of high molecular mass. A considerable improvement in the efficiency of transfer can be achieved by directly digesting the DNA in the gel by DNase I. The method is illustrated in the example of the preferential binding of histone H1 to superhelical plasmid DNA.
Versatile low‐viscosity sieving matrices for nondenaturing DNA separations using capillary array electrophoresis
Authors: Madabhushi Ramakrishna S. , Vainer Marina , Dolnik Vladislav , Enad Shellee , Barker David L. , Harris Dennis W. , Mansfield Elaine S. 
Published: May 2005

AbstractThe high‐resolution separation of double‐stranded DNA (dsDNA) has important applications in physical mapping strategies and in the analysis of polymerase chain reaction (PCR) products. Although high‐resolution separations of dsDNA by capillary electrophoresis (CE) have been reported, pulsed fields were required to achieve complete resolution of DNA fragments beyond 23 kilobase pairs (kbp). Here, we report a single formulation to separate a broad range (80 bp – 40 kbp) of DNA fragments without the use of pulsed fields. We used a low‐viscosity sieving medium (ca. 5 cP, at 25°C) based on polyethyleneoxide (PEO) to separate DNA fragments up to 40 kbp. The matrix contained a mixture of 0.5% PEO (Ma 106) to separate fragments up to 1.5 kbp, combined with 0.1% PEO (Ma 8 × 106) to separate fragments between 1–40 kbp, within a single run. All PEO matrix formulations tested were compatible with a variety of intercalating dyes and with two different capillary wall coating methods. We obtained a detection limit of 25 fg of a 200 bp DNA quantitation standard using Vistra Green in the matrix. Resolution was best using short injection times (5 s or less) and low field strengths (approximately 100 V/cm). Sample runs were complete in 70 min, and use of the capillary array electrophoresis (CAE) system permitted high‐throughput DNA analysis. The size range separated is approximately 10 times greater than with conventional slab gel separations.
Measurement of antibody‐antigen dissociation constants using fast capillary electrophoresis with laser‐induced fluorescence detection
Authors: Tao Li , Kennedy Robert T. 
Published: May 2005

AbstractThe dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate (FITC)‐labeled insulin and unlabeled insulins from several species were measured using capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF). Kd determinations were made by separating free FITC‐insulin and its complex with the antibody in equilibrated solutions in 6 s or less. The use of LIF detection allowed quantification of free and bound FITC‐insulin in the picomolar range, as is required to measure Kd's below 1 nM. The Kd of FITC‐insulin with the antibody was determined to be 0.25 nM by Scatchard analysis. The Kd's of the antibody with unlabeled insulins from several species were obtained by fitting bound over free FITC‐insulin as a function of unlabeled insulin concentration data from a series of solutions containing a fixed concentration of FITC‐insulin and antibody and variable concentrations of insulin to the expected curve derived from the equilibria and mass balance of the solutions. Kd's for the different insulins were between 0.34 and 0.64 nM.
Electrophoretic demonstration of high molecular weight fibrin degradation products persisting in chronic subdural hematomas
Authors: Toyosawa Mikiko , Kashiwagi Shiro , Pei Wang , Fujisawa Hirosuke , Ito Haruhide , Nakamura Kazuyuki 
Published: May 2005

AbstractLocal hyperfibrinolysis plays an important role in the pathogenesis of chronic subdural hematoma (CSH). The purpose of this study is to elucidate the nature of the local hyperfibrinolysis in CSH, by comparing the pattern of fibrin/fibrinogen degradation product (FDP) of hematomas with that of purified fibrin clots digested by plasmin in vitro. Forty‐seven hematoma samples were subjected to the analysis. FDPs of CSH and the purified fibrin clots digested by plasmin for different incubation times were identified using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting, and then quantified by densitometry. The effect of alpha‐2 plasmin inhibitor (A2‐PI) on the degradation process was also studied. The FDP pattern of CSH was similar to those of purified fibrin digested by plasmin for 0.75–2h. The FDP composition of CSH was closest to that of purified fibrin digested for 1h. By adding the physiological concentration of A2‐PI at the second hour, further degradation of high molecular weight FDPs was inhibited and the early FDP pattern persisted after 24h. Local hyperfibrinolysis in CSHs is characterized by incomplete fibrinolysis, which occurs only on the solid fibrin clot, and is arrested in the liquid hematoma. As a result, high molecular weight FDPs persist in CSHs for weeks or months in the hematoma. A2‐PI seems to play an important role in producing this unique FDP pattern in CSH.
Two‐dimensional electrophoresis of membrane proteins: A current challenge for immobilized pH gradients
Authors: Adessi Céline , Miege Christine , Albrieux Catherine , Rabilloud Thierry 
Published: May 2005

AbstractMembrane proteins were separated by high resolution two‐dimensional (2‐D) electrophoresis. On isoelectric focusing (IEF) with immobilized pH gradients severe protein losses in the resulting 2‐D map were observed when compared with carrier ampholyte‐based IEF. This has been noticed for two different biological systems, namely the chloroplast envelope of spinach and the endocytic vesicles from Dictyostelium discoideum. The possible mechanisms of these losses on immobilized pH gradients are discussed.
Low molecular weight proteins secreted by peritoneal macrophages obtained from 2,3,7,8‐tetrabromodibenzo‐p‐dioxin‐treated NMRI mice
Authors: Schlatterer Kathrin , Esmaeli Anoosh , Chandra Angelika , Hofmann Dieter , Lerch Anja , Schlatterer Bert , Demirhan Ilhan , Chandra Prakash 
Published: May 2005

AbstractThe protein secretion patterns in a macrophage‐like cell line (CBrD), established from the peritoneal cells of NMRI mice treated with the dioxin analog 2,3,7,8‐tetrabromodibenzo‐p‐dioxin (TBrDD), were analyzed by high resolution two‐dimensional gel electrophoresis (2‐D PAGE), and compared to the pattern of proteins secreted by control macrophages which were intraperitoneally activated by bacterial lipopolysacchride. The most striking alterations were observed in the low molecular range. The transformed cells encode a number of low molecular mass proteins (10–20 kDa) which were not detected in control cells under identical experimental conditions. The protein pattern with respect to isoelectric point, molecular weight, optical density (OD) and area of the spot (in mm2) has been depicted by computer analysis in relation to a standardized spot outline and the spot's background (in OD). It is concluded that the transformation of murine peritoneal macrophages by TBrDD leads to an upregulation of proteins, in particular of low‐molecular‐weight proteins.
Mapping and identification of Brucella melitensis proteins by two‐dimensional electrophoresis and microsequencing
Authors: Teixeira‐Gomes Ana P. , Cloeckaert Axel , Bézard Guy , Dubray Gérard , Zygmunt Michel S. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) gel electrophoresis was used to map Brucella melitensis proteins. The 2‐D proteins map of B. melitensis B115 revealed 595 silverstained protein spots separated by both isoelectric point and molecular mass. Twenty‐five proteins were identified either by immunoblotting using monoclonal antibodies (MAbs) or by N‐terminal microsequencing. The protein spots identified by MAbs were the 89 kDa outer membrane protein, DnaK, bacterioferritin, CP24, and BP26. Some spots were identified by N‐terminal microsequencing as proteins whose sequences had been reported previously from Brucella, such as three heat‐shock proteins, namely DnaK, GroEL and GroES; bacterioferritin; Cu‐Zn superoxide dismutase; and the 50S ribosomal protein L7/L12. Other proteins had amino acid sequences homologous with those of various proteins from other bacteria found in protein databases: ClpP; the 10K‐S protein; the ORFU phosphoprotein; succinyl‐CoA synthetase alpha subunit; an inorganic pyrophosphatase; the Fe and/or Mn superoxide dismutase; the nucleoside diphosphate kinase, an amino acid ABC type transporter, and an electron transfer flavoprotein small subunit. Seven proteins were identified with N‐terminal sequences not yet reported in databases. The 2‐D map established in this study will be the basis for comparative studies of protein expression in Brucella.
Electrophoretic analysis of cyanide depletion by Pseudomonas alcaligenes
Authors: Zaugg Spencer E. , Davidson Robert A. , Walker Jeffrey C. , Walker Edward B. 
Published: May 2005

AbstractBacterial‐facilitated depletion of cyanide is under development for remediation of heap leach operations in the gold mining industry. Capillary electrophoresis was found to be a powerful tool for quantifying cyanide depletion. Changes in cyanide concentration in aqueous suspensions of Pseudomonas alcaligenes bacteria and cyanide at eleveated pH were easily monitored by capillary electrophoresis. The resulting data can be used to study rates of cyanide depletion by this strain of bacteria. Concentrations of these bacteria at 105 cells/mL were found to reduce cyanide from 100 ppm to less than 8 ppm in four days. In addition, other ions of interest in cyanide metabolism, such as formate, can be simultaneously analyzed. Direct UV detection of cyanide at 192 nm further simplifies the analytical method for these ions.
Capillary zone electrophoresis of nitrophenols with off‐line isotachophoretic sample pretreatment
Authors: Kaniansky Dušan , Krčmová Eva , Madajová Vlasta , Masár Marián 
Published: May 2005

AbstractPreparative capillary isotachophoresis (ITP) operating in a discontinuous frationation mode was studied as a sample pretreatment technique for capillary zone electrophoresis (CZE) trace analysis of a group of ten nitrophenols. Different separation mechanisms employed by these capillary electrophoresis techniques made possible a group isolation of the studied analytes by ITP while their CZE resolutions based on differences in the interactions with polyvinylpyrrolidone (PVP) provided suitable conditions for a final analytical evaluation. Experiments with model and practical water samples revealed high and reproducible recovery rates of the ITP pretreatment for nitrophenols when sample loadability limits of the preparative ITP equipment were met. A main disadvantage of the studied ITP‐CZE combination was an inefficient use of the pretreated sample as only 0.5% of the fraction containing nitrophenols was used in the final CZE step. Despite these limitations associated with the CZE sample injection, the concentration limit of detection (LOD) for nitrophenols in practical water samples could be reduced to 2–8 ppb concentrations (photometric absorbance detector operating at a 254 nm wavelength) when 200 μL sample volumes were taken for the ITP pretreatment. This was accompanied by an efficient sample clean‐up as no other trace ionic constituents originating from the samples were detected on CZE.
Determination of ethylenediaminetetraacetic acid as the nickel chelate in environmental water by solid‐phase extraction and capillary electrophoresis/tandem mass spectrometry
Authors: Sheppard Robin L. , Henion Jack 
Published: May 2005

AbstractAn automated extraction procedure using solid‐phase extraction (SPE) disk technology was developed for the qualitative and quantitative determination of ethylenediaminetetraacetic acid (EDTA) in water samples. The procedure involves conversion of all free and chelated EDTA present into the nickel EDTA chelate followed by extraction on strong anion exchange extraction disks. A 5 mL water sample is extracted and concentrated to 30 μL. This extract is then analyzed by capillary electrophoresis (CE) using ion spraytandem mass spectrometry (MS) for selective detection. An amine‐coated capillary column is used to separate anions using negative voltage and high electroosmotic flow. A self‐aligning liquid junction is used as the CE/MS interface. The sample is injected using field amplification for enhanced concentration detection limits down to 0.15 μg/L. This detection limit is about five times lower than any report we have found in the literature and provides the unique specificity afforded by mass spectrometry. Total sample preparation and run time was forty minutes per sample, including evaporation steps and capillary rinsing between runs. A tray of 24 vials, including samples and standards, was analyzed for these experiments and was easily finished within one day. These experiments demonstrate that CE/MS can be a rugged, quantitative technique, which, with proper sample preparation and focusing techniques, can compete with the concentration sensitivity of established analytical methods.
Very alkaline immobilized pH gradients for two‐dimensional electrophoresis of ribosomal and nuclear proteins
Authors: Görg Angelika , Obermaier Christian , Boguth Günther , Csordas Adam , Diaz Jean‐Jacques , Madjar Jean‐Jacques 
Published: May 2005

AbstractBasic proteins normally lost by the cathodic drift of carrier ampholyte focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two‐dimensional (2‐D) electrophoresis under equilibrium conditions using immobilized pH gradients (IPGs) 4–10 and 6–10 using a previously published protocol (Görg et al., Electrophoresis 1988, 9, 531–546). In the present study we have extended the pH gradient to pH 12 with IPGs 8–12, 9–12 and 10–12 for the analysis of very basic proteins. Different optimization steps with respect to pH engineering, gel composition and running conditions, such as substitution of acrylamide by dimethylacrylamide and addition of isopropanol with and without methylcellulose to the IPG rehydration solution (in order to suppress the reverse electroosmotic flow) were necessary to obtain highly reproducible 2‐D patterns of ribosomal proteins from HeLa cells and mouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under steady‐state conditions. Due to the selectivity of isoelectric focusing in IPG 9–12, where the more acidic proteins abandon the gel, the tedious procedure of nuclei preparation prior to histone extraction can be omitted.
Towards design and comparison of World Wide Web‐accessible myocardial two‐dimensional gel electrophoresis protein databases
Authors: Plei Klaus‐Peter , Sander Stefan , Oswald Helmut , Regitz‐Zagrosek Vera , Fleck Eckart 
Published: May 2005

AbstractIn addition to the recently published HEART‐2DPAGE – a myocardial World Wide Web‐accessible 2‐DE gel protein database – the usage and installation of software tools are described with regard to the hard‐ and software environments. Further, access to the HEART‐2DPAGE from other two‐dimensional electrophoresis (2‐DE) databases using name or accession code of a protein is now available. Moreover, database images, published in the myocardial HSC‐2DPAGE and HEART‐2DPAGE databases are compared. Using the warping tool of the common image processing system Khoros the database images are matched and added in order to visualize the effects of warping. The application of such image processing tools is aimed at improving the comparability of protein spot patterns of different gel images available through the net.
The assay development of a molecular marker for transmissible spongiform encephalopathies
Authors: Lee Kelvin H. , Harrington Michael G. 
Published: May 2005

AbstractThe transmissible spongiform encephalopathies are a group of neurodegenerative diseases which include Creutzfeldt‐Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. Two‐dimensional electrophoresis of proteins was previously used to identify two marker proteins, 130/131, which are selectively present in the cerebrospinal fluid (CSF) of patients with CJD and not in patients with other dementias. The recent characterization of these proteins by amino acid sequencing has identified them as members of the 14‐3‐3 family of proteins. Polyclonal antibodies against 14‐3‐3 (all isoforms), 14‐3‐3γ, 14‐3‐3β, and 14‐3‐3θ are immunoreactive with a 30 kDa marker band from CJD CSF. Silver staining of two‐dimensional electrophoresis separated BSE CSF proteins does not identify a similar marker. However, 14‐3‐3 immunoreactivity is found in cattle CSF when these proteins are blotted to polyvinylidene difluoride but not when blotted to nitrocellulose.