bluuebunny/crossref_metadata_2025_split huggingface dataset:
“Schizophrenic” behavior of zones in capillary zone electrophoresis: Explanation of an old problem
Authors: Gebauer Petr , Desiderio Claudia , Fanali Salvatore , Boček Petr 
Published: May 2005

AbstractBackground electrolyte (BGE) systems with two coions are frequently used in capillary zone electrophoresis (CZE), especially in cases where indirect optical detection is employed. This study investigates the behavior of analytes, which possess mobilities intermediate to those of the BGE coions used. Besides the expected behavior, where the analytes provide either tailing or fronting zones, unusual behavior with extraordinary zone broadening is also observed in some cases. The explanation for this effect is that binary BGE systems involve, as a physico‐chemical rule, a region where the analytes are forced by one coion to give tailing zones and simultaneously by the other coion to give fronting zones. The result of this “schizophrenic” situation is extraordinary zone broadening and deterioration of the detection record. A series of experiments is presented showing in a telling way the electromigration behavior of the discussed type of zones as well as the ways to remedy the deterioration of the peak shape by a mere slight changing of the quantitative composition of the BGE used.
Detectability improvements in capillary zone electrophoresis by combining single capillary isotachophoretic preconcentration and frequency doubled argon ion laser‐induced fluorescence detection
Authors: Enlund Anna Maria , Schmidt Staffan , Westerlund Douglas 
Published: May 2005

AbstractDue to the small path length and low injection volume the concentration limit of detection is comparatively poor in capillary electrophoresis (CZE) with UV detection. This limitation can be overcome by means of preconcentration methods and/or improved detection techniques. This paper describes a strategy where isotachophoresis (ITP) is used to preconcentrate a new cholinesterase inhibitor (NXX‐066) prior to a capillary zone electrophoresis analysis in the same single capillary. A hydrodynamic backpressure is used to prevent the analyte from migrating out of the capillary. Laser‐induced fluorescence (LIF) is used to further increase the detectability. The total gain in detectability with ITP‐CZE‐LIF compared to CZE‐UV was at least 5500‐fold, and it is possible to determine NXX‐066 at the 1 nM level. The ITP‐CZE method was further evaluated for two β‐blockers; the mean coefficient of variation of the peak areas was 3.4% and the linearity of the calibration plots was satisfying.
Population genetic study of four short tandem repeat loci in the Belgian population, using capillary electrophoresis
Authors: Van Hoofstat David E. O. , Deforce Dieter L. D. , Millecamps Rebecca E. M. , Brochez Veerle M. , Van Geldre Els G. L. , Vergauwe Annemieke , Sijs Annick , Van Den Eeckhout Elfriede G. 
Published: May 2005

AbstractAllele frequencies of four short tandem repeat loci (HumCD4, HumTH01, HumD21S11 and HumSE33) were investigated in a sample of 395 unrelated Belgian individuals using multiplex polymerase chain reaction and capillary electrophoresis. Automated laser fluorescence was used to detect four fluorescent dyes, enabling the use of an internal standard within each lane. With this method rapid typing with high resolution was obtained and the different alleles were grouped on a statistical base. All loci meet Hardy‐Weinberg expectations. The allelic frequency data, together with the constructed allelic ladder, can be used in paternity testing and personal identification in the medical and forensic sciences.
Two replica blotting methods for fast immunological analysis of common proteins in two‐dimensional electrophoresis
Authors: Neumann Holger , Müllner Stefan 
Published: May 2005

AbstractThe combination of two‐dimensional electrophoresis (2‐DE) and subsequent Western blot analysis with antibodies directed against common cellular proteins is a straightforward and reliable method to quickly generate fix points in a protein map. In order to assure high accuracy in the allocation of protein spots, two different replica blotting methods for semidry blotting devices were established. The first of the two was described by Johansson (Electrophoresis 1987, 8, 379–383). By systematically changing the direction of the blotting current, proteins were simultaneously transferred from one gel onto two membranes placed at both sides of the gel. However, several modifications of this method were necessary in order to use a semidry blotting device. The second method described here combines the standard blotting procedure with the generation of a ‘contact copy’ from the gel. Both systems offer the possibility to subject one membrane to antibody‐mediated imaging, while the second membrane can be stained with highly sensitive total protein detection procedures. Protein identification is then carried out by comparing the signals on both matrices.
Two‐dimensional electrophoretic analysis of mixed lineage kinase 2 N‐terminal domain binding proteins
Authors: Rasmussen Richele K. , Ji Hong , Eddes James S. , Moritz Robert L. , Reid Gavin E. , Simpson Richard J. , Dorow Donna S. 
Published: May 2005

AbstractThe mixed lineage kinase 2 (MLK2) protein contains several structurally distinct domains including an src homology (SH) 3 domain, a kinase catalytic domain, two leucine zippers, a basic motif and a cdc42/rac interactive binding motif. These domains have been recognized mainly for their involvement in protein‐protein interactions in signal transduction networks. The SH3 domain in particular has been implicated in control of signaling events. To identify proteins that interact with MLK2, the N‐terminal 100 amino acids, including the SH3 domain, were expressed as a glutathione S‐transferase (GST) fusion protein. This fusion protein (MLK2N) was used as an affinity ligand to isolate binding proteins from lysates of 35S‐radiolabeled MDA‐MB231 breast carcinoma cells. When the radiolabeled binding proteins were subjected to 2‐DE, proteins of Mr 55 000, 31 500 and 34 000 bound consistently to the MLK2N domain fusion protein, but not to the GST control. Two of the binding proteins were isolated from whole cell lysates by preparative 2‐DE and subjected to in‐gel digestion and capillary or microbore reverse‐phase high performance liquid chromatography (RP‐HPLC). Resultant peptides were analyzed by peptide masss fingerprinting, N‐terminal Edman degradation or tandem mass spectrometry. The 55 000 protein was identified as the cytoskeletal protein, β‐tubulin, and this was verified by immunoblotting of proteins in the MLK2N binding fraction with anti‐tubulin antibodies. The 31 500 protein has been identified as prohibitin, a protein that has been implicated in both signal transduction and cell cycle arrest.
Extraction of membrane proteins by differential solubilization for separation using two‐dimensional gel electrophoresis
Authors: Molloy Mark P. , Herbert Ben R. , Walsh Bradley J. , Tyler Margaret I. , Traini Mathew , Sanchez Jean‐Charles , Hochstrasser Denis F. , Williams Keith L. , Gooley Andrew A. 
Published: May 2005

AbstractWe describe the extraction and enrichment of membrane proteins for separation by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three‐step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris‐base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N‐terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2‐D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2‐D PAGE using broad‐range pH 3.5–10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.
Development of mini‐gel technology in two‐dimensional electrophoresis for mass‐screening of samples: Application to tears
Authors: Glasson Melissa J. , Molloy Mark P. , Walsh Bradley J. , Willcox Mark D. P. , Morris Carol A. , Williams Keith L. 
Published: May 2005

AbstractDespite the extensive literature available on tear proteins and lipids, very little has been reported on the tear fluid as a whole and it's changes in contact lens wear or ocular diseased patients. Initially a human reflex tear two‐dimensional map was created by Molloy et al. (Electrophoresis 1997, 18, 2811–2815), using this information a process for mass‐screeniung was established. The large format two‐dimensional technique was evaluated, using a basal tear reference map, and modified to describe a fast, efficient and cost effective method of protein separation. The use of one pH 3–10 18 cm nonlinear immobilised pH gradient (IPG) strip and two mini‐gels for the second‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) results in an effective separation of tear proteins which will be applied in diagnostic studies of tear samples.
Optimization of capillary chromatography ion trap‐mass spectrometry for identification of gel‐separated proteins
Authors: Courchesne Paul L. , Jones Michael D. , Robinson John H. , Spahr Chris S. , McCracken Susan , Bentley David L. , Luethy Roland , Patterson Scott D. 
Published: May 2005

AbstractThe current paradigm for protein identification using mass spectrometric derived peptide‐mass and fragment‐ion data employs computer algorithms which match uninterpreted or partially interpreted fragment‐ion data to sequence databases, both protein and translated nucleotide sequence databases. Nucleotide sequence databases continue to grow at a rapid rate for some species, providing an unsurpassed resource for protein identification in those species. Ion‐trap mass spectrometers with their ability to rapidly generate fragment‐ion spectra in a data‐dependent manner with high sensitivity and accuracy has led to their increased use for protein identification. We have investigated various parameters on a commercial ion trap‐mass spectrometer to enhance our ability to identify peptides separated by capillary reversed phase‐high performance liquid chromatography (RP‐HPLC) coupled on‐line to the mass spectrometer. By systematically evaluating the standard parameters (ion injection time and number of microscans) together with selection of multiple ions from the full mass range, improved tandem mass spectrometry (MS/MS) spectra were generated, facilitating identification of proteins at a low pmol level. Application of this technology to the identification of a standard protein and an unknown from an affinity‐enriched mixture are shown.
Enantiomeric separation of methadone by cyclodextrinbased capillary and recycling isotachophoresis
Authors: Lanz Matthias , Caslavska Jitka , Thormann Wolfgang 
Published: May 2005

AbstractThe separation of methadone enantiomers by cationic capillary isotachophoresis (CITP) and recycling isotachophoresis (RITP) having (2‐hydroxypropyl)‐β‐cyclodextrin (OHP‐β‐CD) as chiral selector in the leading electrolyte is described. Sodium acetate/acetic acid (pH between 4 and 5) served as leading electrolyte (catholyte) and acetic acid as terminator (anolyte). Complete separation of the enantiomers was obtained by CITP in a 50 μm internal diameter (ID) fused‐silica capillary and in a 500 μm ID Teflon capillary. In the first approach, enantiomeric separation could be monitored via UV absorbance detection at low wavelength. With the second instrumental setup, an additional conductivity sensor permitted the visualization of the enantiomeric separation and the characterization of the buffer system employed. A 10 mM sodium acetate/acetic acid leading buffer of pH 4.3, containing 5 mM OHP‐β‐CD, was found to provide best enantiomeric separation and was thus chosen for RITP. With RITP processing of a few mg of racemic methadone, partial separation of methadone enantiomers was obtained. R‐(−)‐methadone and S‐(+)‐methadone were found to be significantly (up to about 80%) enriched at the front and back side, respectively, of the isotachophoretic zone. The enantiomeric composition of methadone in the collected fractions was assessed by chiral capillary zone electrophoresis (CZE) and circular dichroism spectroscopy. CZE was found to represent a simple and efficient method for the determination of the enantiomeric excess, whereas the latter technology was noted to be the superior approach for properly characterizing fractions that contain similar amounts of the two enantiomers. Furthermore, chiral RITP and analysis of the collected fractions by circular dichroism spectroscopy is shown to be potentially useful for identification of single enantiomers in absence of pure chiral standards.
Application of free‐flow electrophoresis for isolation and purification of proteins and peptides
Authors: Loseva Olga I. , Gavryushkin Alexander V. , Osipov Vladimir V. , Vanyakin Evgeny N. 
Published: May 2005

AbstractFree‐flow electrophoresis (FFE) has been applied to the separation and purification of a variety of proteins and polypeptides: bee venom, tumor necrosis factor, interleukin‐1β, interferon‐α and superoxide dismutase. FFE at constant pH and conductivity of the carrying buffer is shown to be efficient at various separation schemes. In some cases, the method allows us to obtain proteins with a purity of more than 90% at a productivity of 20–30 mg/h. An electrophoretic apparatus with a new, multi‐sectional construction of the electrophoretic chamber and a system for cross‐displacement of carrying buffer in the chamber is described.
Free‐flow electrophoresis for fractionation of Arabidopsis thaliana membranes
Authors: Bardy Nicolas , Carrasco Antoine , Galaud Jean‐Philippe , Pont‐Lezica Rafael , Canut Hervé 
Published: May 2005

AbstractHighly purified tonoplast and plasma membrane vesicles were isolated from microsomes of Arabidopsis thaliana by preparative free‐flow electrophoresis. The most electronegative fractions were identified as tonoplast using nitrate‐inhibited Mg2+‐ATPase as enzyme marker. The least electronegative fractions were identified as plasma membrane using glucan‐synthase II, UDPG: sterolglucosyl‐transferase, and vanadate‐inhibited Mg2+‐ATPase as enzyme markers. Other membrane markers, latent inosine‐5′‐diphosphatase (Golgi), NADPH‐cytochrome‐c reductase (endoplasmic reticulum) and cytochrome‐c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane. Immunoblot analysis of membrane fractions by antibodies directed against tonoplast and plasma membrane proteins confirmed the nature and the purity of the isolated membranes. The cytoskeletal protein actin, which was also identified by immunoblotting, was found to be specifically attached to the plasma membrane vesicles. The structural and functional integrity of the isolated membranes from Arabidopsis thaliana is discussed in the light of results obtained for the location of receptors and enzymes, or for the determination of ligand binding activity.
Immuno‐isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting
Authors: Lüers Georg H. , Hartig Roland , Mohr Heribert , Hausmann Michael , Fahimi H. Dariush , Cremer Christoph , Völkl Alfred 
Published: May 2005

AbstractImmuno‐isolation is a powerful technique for the isolation of cells as well as subcellular organelle populations based on their antigenic properties. We have established a method for immuno‐isolation of peroxisomes (PO) from both rat liver and the human hepatoblastoma cell line HepG2 using magnetic beads as solid support. A polyclonal antibody raised against the cytoplasmic C‐terminal 10 amino acids of the rat 70 kDa peroxisomal membrane protein was covalently bound to magnetic beads (Dynabeads M‐450). The coated beads were incubated with a light mitochondrial fraction and the organelle‐bead complexes formed were separated by magnetic sorting in a free‐flow system without pelleting the complexes during the isolation procedure. Scanning electron microscopy revealed decoration of beads with particles measuring 150–400 nm in diameter. The particles were identified as PO by catalase cytochemistry and biochemically by marker enzyme analysis, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as well as immunoblotting for specific detection of peroxisomal matrix, core and membrane proteins. The functional significance of PO in man is emphasized by the existence of inherited diseases such as the Zellweger syndrome in which intact PO are lacking, but peroxisomal remnants called “ghosts” are observed instead. Peroxisomal disorders are usually studied using skin fibroblast cell lines derived from afflicted patients and immuno‐magnetic separation may prove particularly useful for the investigation of such cultured cells and for further elucidation of the pathogenesis of fatal peroxisomal disorders.
Electrophoretic analysis of multiple protein‐DNA interactions
Authors: Fried Michael G. , Daugherty Margaret A. 
Published: May 2005

AbstractUnder favorable conditions, native gel electrophoresis allows the resolution of protein‐DNA complexes that differ in stoichiometry, identities of occupied DNA sequences (configuration), and macromolecular conformation. This technique provides a unique opportunity to analyze, in thermodynamic terms, the molecular interactions that govern the equilibrium distributions of species in protein‐DNA mixtures. Here we describe a general theoretical approach to the analysis of electrophoretic band intensities, and provide examples of its application to the analysis of several interacting systems.
Effects of Alu insertions on gene function
Authors: Szmulewicz Martin N. , Novick Gabriel E. , Herrera Rene J. 
Published: May 2005

AbstractAlu elements are a family of short interspersed repetitive elements (SINEs) found exclusively in primates. These elements are around 300 base pairs long, are found in excess of one million copies per diploid genome, and are dispersed throughout the human genome. Alu elements are scattered by a mechanism called “retrotransposition”. Three independent steps are involved in retrotransposition: transcription of the Alu repetitive element, reverse transcription of the Alu RNA and integration of the Alu cDNA. The fact that Alu elements retrotranspose so readily suggests that they have a myriad of effects on the genome, mostly by inactivating genes or altering their function. These characteristics of Alu repetitive elements point to these repetitive DNA fragments as a major driving force for evolution. In addition, Alu elements are known to adopt diverse functions depending on the context of the surrounding genetic material into which they insert. In this article, we review some of the evidence that demonstrates the functional significance of Alu repeats.
The detection of oligonucleotide N3′ → P5′ phosphoramidate/RNA duplexes with capillary gel electrophoresis
Authors: Dedionisio Lawrence , Raible Annette M. , Gryaznov Sergei M. 
Published: May 2005

AbstractOligonucleotide N3′ → P5′ phosphoramidates (3′‐phosphoramidates) are DNA analogs that are presently under investigation as potential therapeutic agents. These compounds may also hold promise as a diagnostic tool. Here, we describe a rapid method for the analysis of single‐stranded RNA fragments utilizing 3′‐phosphoramidate oligonucleotides as probes in conjunction with capillary gel electrophoresis (CGE). 3′‐Phosphoramidate 9‐mers were mixed with complimentary RNA, and CGE was used to monitor duplex formation. Complimentary strands of RNA and 3′‐phosphoramidate formed duplexes that gave unique relative mobilities based on an internal standard. The ability of CGE to discriminate between perfect duplexes and duplexes that contain a base mismatch was also investigated. However, the primary focus of this work was to determine CGE's ability to detect the presence of the 3′‐phosphoramidates/RNA duplex under routine electrophoretic running conditions. Polyacrylamide gel electrophoresis analysis was utilized to verify duplex formation.
An alternative for purification of low soluble recombinant hepatitis C virus core protein: Preparative two‐dimensional electrophoresis
Authors: Yvon Stéphane , Rolland Dominique , Charrier Jean‐Philippe , Jolivet Michel 
Published: May 2005

AbstractFor isolation of low soluble recombinant full‐length (amino acids 1–191) core protein of hepatitis C virus (HCV) overexpressed in Escherichia coli, the advantage of combining two electrophoretic techniques, in comparison with chromatographic separation, is demonstrated. The protein extract was first solubilized in agents compatible with electrophoretic separation. Using preparative liquid phase isoelectric focusing (IEF) the protein of interest was first concentrated within a defined acidic pH range. These fractions were then submitted to preparative sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis (SDS‐PAGE) to isolate the 22 kDa protein. The second‐dimensional step allowed the isolation of 2 mg of the purified recombinant HCV core protein (rHCV‐C191) from 1.5 g bacterial pellet. This quantity is sufficient to characterize the protein and to perform immunogenicity studies. This procedure of two‐dimensional preparative electrophoresis is applicable to a wide range of biological samples and represents an alternative for purification of insoluble proteins.
Subtyping of transferrin by isoelectric focusing in immobilized pH gradients
Authors: Guo Yaojun , Li Xinhui , Zhu Haizhen , Shen Ziwei 
Published: May 2005

AbstractIsoelectric focusing in immobilized pH gradients ranging from pH 5.05 to 5.60 was used to study the distribution of transferrin (Tf) subtypes and their gene frequencies from 188 unrelated healthy donors of the Han population in Beijing. Six phenotypes (TfCl, TfC2, TfC1C2, TfC1C3, TfC1Dchi and TfC2Dchi) were detected and Tf*C3 has never before been reported in the Han population. Gene frequencies were as follows: Tf*Cl = 0.7420, Tf*C2 = 0.2420, Tf*C3 = 0.0027, Tf*Dchi = 0.0133. There is good agreement between the observed and expected values corresponding to the Hardy‐Weinberg equilibrium (∑χ2 = 0.9183, df = 2, 0.5 < P, < 0.75). The allele frequencies for Tf*C1, Tf*C2 and Tf*Dchi agreed with those previously reported for the Chinese Han population.