bluuebunny/crossref_metadata_2025 huggingface dataset:
A diagnostic test for scrapie‐infected sheep using a capillary electrophoresis immunoassay with fluorescent‐labeled peptides
Authors: Schmerr Mary Jo , Jenny Allen
Published: May 2005

Scrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod‐shaped fibrils in the brain that form from an aggregated protein. This protein (PrPSC) is a protease‐resistant form of a normal host cell protein. When the aggregated protein is denatured in sodium dodecyl sulfate (SDS) and β‐mercaptoethanol, a monomer form of ∼27 kDa molecular mass is observed. A competition immunoassay to detect PrPSCfrom scrapie‐infected sheep was developed using free zone capillary electrophoresis with laser‐induced fluorescence (LIF) for detection and flourecein‐labeled sysnthetic peptides from PrPSC. Antibodies were made to each respective peptide and used in the competition assay. The fluorescent‐labeled peptides bound to the antibody were separated from the unbound peptides using 200 mMTricine, pH 8.0, containing 0.1%n‐octylglucoside and 0.1% bovine serum albumin (BSA). The amount of antibody that would bind ∼50% of the fluorescent‐labeled peptide was determined for each peptide. When unlabeled peptide was added to the assay, ∼2 fmoles of the peptide could be measured. When PrPSCextracted from infected sheep brain was added to the assay, approximately 135 pg of PrPSCcould be detected. When preparations from normal sheep were assayed, there was little or no competition for the bound peptides. Assays using two of the peptides, peptides spanning amino acid positions 142–154 and 155–178, clearly differentiated scrapie‐positive sheep from normal animals. This assay is a new method that can be used to diagnose scrapie and, possibly, other transmissible spongiform encephalopathies in animals and in humans.
Exploitation of a monoclonal antibody for weak affinity‐based separation in capillary gel electrophoresis
Authors: Ljungberg Helén , Ohlson Sten , Nilsson Staffan
Published: May 2005

Weak biospecific recognition has been established for affinity separation in high performance liquid chromatography (HPLC). The use of weak affinity chromatography (WAC) has been limited previously by the insufficient separation efficiency achieved, allowing only some 1000 plates/m to be obtained. However, it has been shown that chiral drug separation can be performed with capillary affinity gel electrophoresis (CAGE) at considerably improved efficiency as compared with traditional chromatographic procedures. Our present study demonstrates the potential of weak affinity monoclonal antibodies as a generic method for immunologically based separations in capillary electrophoresis. Monoclonal antibodies were polymerized within a silica capillary and were used for the separation of structurally similar carbohydrate antigens. The results indicate that weak biospecific interactions can be utilized in a CAGE format to produce highly selective separation of the α‐ and β‐forms of p‐nitro‐phenyl‐labeled maltose. It remains to be seen, however, how efficient weak affinity separation in CAGE can be compared with affinity HPLC protocols. Details of typical separations and of the preparation of the antibody gel are presented.
Genome encyclopedias and their use for comparative analysis of Rhodobacter capsulatus strains
Authors: Fonstein Michael , Nikolskaya Tatiana , Kogan Yakov , Haselkorn Robert
Published: May 2005

This paper consists of two components: the use of gene encyclopedias in genomic studies and Rhodobacter capsulatus genome project. A survey of vectors used for encyclopedia construction includes a brief discussion of their relative advantages and limitations. Projects employing various methods of encyclopedia assembly including the comparison of restriction patterns, restriction maps, linking by hybridization, oligonucleotide fingerprinting, sequence tagged site (STS) fingerprinting and encyclopedias derived from genetic maps are listed and briefly described. The R. capsulatus SB 1003 genome project started with the construction of its cosmid encyclopedia, which comprises 192 cosmids covering the chromosome and the 134 kbp plasmid in strain SB 1003, with the exact map coordinates of each cosmid. In a pilot sequencing study, several cosmids were individually subcloned using the vector M13mp18 and merged into one 189 kbp contig. About 160 open reading frames (ORFs) identified by the CodonUse program were subjected to similarity searches. The biological functions of eighty ORFs could be assigned reliably using the WIT (what is there) genome investigation environment. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Recently, another 1.2 Mbp genome fragment of the Rhodobacter genome was sequenced using a slightly modified approach. These results together with some genome investigation tools, have been placed at our web site (http://capsulapedia.uchicago.edu). The sequence of R. capsulatus is expected to be completed by summer 1998. A project to construct a systematic set of deletion strains of R. capsulatus in order to assign functions to unknown ORFs has been started. Preliminary data demonstrate the extreme convenience of the unique gene transfer agent (GTA) system to perform such work.
Selective generation of chromosomal cosmid libraries within the Trypanosoma cruzi genome project
Authors: Frohme Marcus , Hanke Jens , Åslund Lena , Pettersson Ulf , Hoheisel Jörg D.
Published: May 2005

From a total genomic cosmid library of the pathogen Trypanosoma cruzi, specific sublibraries of the smallest four chromosomes were isolated by hybridization of the respective chromosomal bands obtained from pulsed‐field gels. These libraries form the basis for initial mapping analyses that should provide information useful for both the ongoing physical mapping of the entire genome and eventual sequence analyses. Selectivity of the procedure was high with 75% to 92%, although cross‐hybridization had to be expected from ubiquitous DNA features, such as centromeric and telomeric sequences, and other regions homologous between individual chromosomes. Overall, the number of identified clones was slightly higher than expected but well within the intrinsic experimental variation considering the uncertainty about the exact genome size, the variability in clonability and the higher frequency of repeat sequences in larger chromosomes. Chromosome III‐ and IV‐specific cosmids were analyzed on Southern blots of chromosomal separations. For strain CL Brener, all clones tested exhibited cross‐hybridization to a homologous chromosome larger than 1 Mbp, supporting the assumption of the respective chromosome couple being diploid pairs.
Hybridization mapping ofTrypanosoma cruzichromosomes III and IV
Authors: Hanke Jens , Frohme Marcus , Laurent Jean‐Pierre , Swindle John , Hoheisel Jörg D.
Published: May 2005

As part of theTrypanosomaGenome Initiative launched by the World Health Organization (WHO), a physical clone map ofTrypanosoma cruzichromosomes III and IV was generated to facilitate both DNA sequence analysis of the parasite's genome and the investigation of chromosome organization. Apart from a few genetic markers, anonymous cosmids were taken from chromosomal sublibraries and individually hybridized to filter arrays of the relevant cosmid library. The probe order was determined from the hybridization fingerprint results and used to define a fitting clone order, with few gaps remaining. The results were independently verified by hybridizations to a bacterial artificial chromosome (BAC) library and, in case of chromosome III, restriction mapping. For gap closure, additional experiments on a total cosmid library were carried out. The possible tiling paths consist of 26 clones for chromosome III (610 kbp) and 28 clones for chromosome IV (680 kbp).
Optimization and automation of fluorescence‐based DNA hybridization for high‐throughput clone mapping
Authors: Scholler Patrik , Heber Steffen , Hoheisel Jörg D.
Published: May 2005

Large‐scale hybridization‐based genome mapping projects, such as the production of sequence‐ready physical clone maps, call for robust and cheap DNA labeling techniques that are amenable to automation. We routinely use a high‐throughput protocol based on fluorescence detection. DNA probes are labeled via polymerase chain reaction (PCR) amplification with primers that are digoxigenin‐modified at their 5′ ends. Alternatively, digoxigenin‐labeled dUTP is incorporated in a random hexamer priming reaction. Hybridization takes place in small volumes by sandwiching the probe between filters and plastic sheets. A fluorescence signal is produced by the activity of alkaline phosphatase attached to an anti‐digoxigenin antibody upon the addition of AttoPhosTM substrate. Signals are directly detected with a charge‐coupled device (CCD) camera and scored by an image data analysis system. DNA filters can be reused at least 40 times without loss of data quality. Significant advantages compared to radioactive techniques are the reduced health risk, enabling highly parallel processing; the production of spot signals uniform in size and intensity, which is essential for efficient image analysis; and a cost reduction of about 70%.
Global analysis of genomic texts: The distribution of AGCT tetranucleotides in the Escherichia coli and Bacillus subtilis genomes predicts translational frameshifting and ribosomal hopping in several genes
Authors: Hénaut Alain , Lisacek Frédérique , Nitschké Patrick , Moszer Ivan , Danchin Antoine
Published: May 2005

Present availability of the genomic text of bacteria allows assignment of biological known functions to many genes (typically, half of the genome's gene content). It is now time to try and predict new unexpected functions, using inductive procedures that allow correlating the content of the genomic text to possible biological functions. We show here that analysis of the genomes of Escherichia coli and Bacillus subtilis for the distribution of AGCT motifs predicts that genes exist for which the mRNA molecule can be translated as several different proteins synthesized after ribosomal frameshifting or hopping. Among these genes we found that several coded for the same function in E. coli and B. subtilis. We analyzed in depth the situation of the infB gene (experimentally known to specify synthesis of several proteins differing in their translation starts), the aceF/pdhC gene, the eno gene, and the rplI gene. In addition, genes specific to E. coli were also studied: ompA, ompF and tolA (predicting epigenetic variation that could help escape infection by phages or colicins).
Structural and functional implications of sequence diversity of Pseudomonas aeruginosa genes oriC, ampC and fliC
Authors: Spangenberg Claudia , Montie Thomas C. , Tümmler Burkhard
Published: May 2005

Sequence analysis of three representative gene loci, oriC, ampC and fliC, in 19 Pseudomonas aeruginosa strains revealed a low sequence diversity that does not correlate with the extensive diversity of P. aeruginosa habitats. Single point mutations lead to a mean sequence diversity of 0.40%, 0.38% and 0.59% for oriC, ampC and a‐type fliC, respectively, but of only 0.05% for b‐type flagellin genes. The analyzed genes encode highly conserved functions that are subject to strong selective pressure. The detected nucleotide substitutions of oriC, accumulating in a central 95 bp region, affect neither the putative DnaA binding sites nor the 13 bp direct repeats that presumably provide the sites to openoriC duplex DNA. Even in P. aeruginosa strain DSM 1128, which exhibits an unusually high sequence variability in several analyzed genes, the 9 bp and 13 bp motifs are conserved, reflecting their essential functional role in replication initiation. The two flagellin types, differing by 37–38% in their primary structure, exhibit pronounced structural and functional homology, as shown by alignment of flagellin variants by hydrophobicity index, probability of surface exposure, chain flexibility and antigenicity, and by cross‐reactivity between both proteins using specific antisera. Five nonsynonymous nucleotide substitutions of ampC lead to β‐lactamase variants that differ in recognition and turnover of substrate, as deduced from the three‐dimensional structure of the highly homologous Enterobacter cloacae β‐lactamase and confirmed by inhibition kinetics. The identified point mutations in the three genes are classified as selectively equivalent sequence variants indicating neutral genetic drift as a mechanism of molecular evolution in P. aeruginosa, rather than positive selection.
Chromosomal rearrangements in enteric bacteria (minireview)
Authors: Sanderson Kenneth E. , Liu Shu‐Lin
Published: May 2005

Early genetic studies showed conservation of gene order in the enteric bacteria. Two recent methods using pulsed field gel electrophoresis (PFGE) to determine the physical map of the genome are: (i) partial digestion with the endonuclease I‐CeuI, which digests the DNA of bacteria in the rrn operon for rRNA (ribosomal RNA), thus establishing the “rrn genomic skeleton” (the size in kbp of the intervals between rRNA operons); (ii) analysis of XbaI and B1nI sites within Tn10 insertions in the chromosome. The order of I‐CeuI fragments, which is ABCDEFG in S. typhimurium LT2 and E. coli K‐12, was found to be conserved in most Salmonella species, most of which grow in many hosts (host‐generalists). However, in S. typhi, S. paratyphi C, S. gallinarum, and S. pullorum, species which are host‐specialized, these fragments are rearranged, due to homologous recombination between the rrn operons, resulting in translocations and inversions. Inversions and translocations not involving the rrn operons are seldom detected except for inversions over the TER (termination of replication) region. Additive genetic changes (due to lateral transfer resulting in insertion of nonhomologous DNA) have resulted in “loops” containing blocks of DNA which provide new genes to specific strains, thus driving rapid evolution of new traits.
Leptospira genomics
Authors: Boursaux‐Eude Caroline , Girons Isabelle Saint , Zuerner Richard
Published: May 2005

The bacterial species Leptospira interrogans (sensu stricto) has a complex genome containing two circular chromosomal replicons. Comparative analysis of the larger chromosome reveals a fluid genetic organization with many large rearrangements differentiating two closely related strains. In the present study new genes were identified by partial sequence analysis of randomly cloned fragments of L. interrogans DNA. These genes were localized in regions of the genome by nucleic acid hybridization with DNA fragments separated by pulsed‐field gel electrophoresis. The resulting genetic maps provide improved resolution for each strain and provide evidence for additional chromosomal rearrangements. Insertion elements may be involved in recombination events, as several are near regions of the chromosome that have undergone rearrangement.
European functional analysis network (EUROFAN) and the functional analysis of the Saccharomyces cerevisiae genome (minireview)
Authors: Dujon Bernard
Published: May 2005

Less than two yeras after the sequence of its genome was completed, the baker's yeast, Saccharomyces cerevisiae, is a leading organism in the rapidly growing field of functional genomics. Two thousands novel protein coding genes, nearly all of them “orphans”, have already been disrupted by the coordinated efforts of a large consortium of European laboratories, EUROFAN, and other initiatives. The mutants are submitted to many specialized functional assays, and studies are performed in parallel at the transcriptome and the proteome levels. With a central repository of mutant yeast strains, and a centralized database, EUROFAN lays the foundations for the future of genomics with yeast serving both as a model and a tool.
Preparative isoelectric focusing and preparative electrophoresis of hydrophobic Candida albicans cell wall proteins with in‐line transfer to polyvinylidene difluoride membranes for sequencing
Authors: Masuoka James , Glee Pati M. , Hazen Kevin C.
Published: May 2005

Hydrophobic proteins in the cell wall of the opportunistic fungal pathogen Candida albicans are involved in adhesion of this organism to host tissue and thus play a role in its pathogenicity. The hydrophobic nature of these proteins results in their loss during purification due to adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic proteins in sufficient quantity for sequencing or biochemical analysis. We describe a system that combines preparative isoelectric focusing with continuous elution preparative electrophoresis. The system provides a two‐dimensional protein separation while maintaining protein solubility and minimizing protein loss due to adsorption. In addition, we have added an in‐line transfer of electrophoretic fractions directly to polyvinylidene difluoride (PVDF) membranes, which further reduces both exposure to apparatus surfaces and purification time.
“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

Background 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

Due 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

Allele 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

The 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

The 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

We 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

Despite 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

The 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

The 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

Free‐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

Highly 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

Immuno‐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

Under 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

Alu 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

Oligonucleotide 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

For 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

Isoelectric 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.
Glutathione S‐transferases: Two‐dimensional electrophoretic protein markers of lead exposure
Authors: Witzmann Frank A. , Daggett Daniel A. , Fultz Carla D. , Nelson Shelli A. , Wright Lynda S. , Kornguth Steven E. , Siegel Frank L.
Published: May 2005

Glutathione S‐transferases (GST) are a family of detoxification isoenzymes that catalyze the conjugation of xenobiotics and their metabolites with reduced glutathione. Lead exposure in rats is known to induce GST isoenzymes in the liver and kidney. These changes in expression have potential use as biomarkers of lead exposure. Because two‐dimensional electrophoresis (2‐DE) enables one to analyze both protein abundance changes and chemical changes in protein structure, 2‐DE was used to determine the effect of in vivo lead exposure on GST isoform expression in rat kidney cytosols. Male Sprague‐Dawley rats were exposed to inorganic lead, and proteins were separated by conventional ISO‐DALT and NEPHGE‐DALT techniques and blotted for immunological identification. Lead exposure caused detectable inductions in both GSTP1 and GSTM1 and quantifiable charge modification in GSTP1. These preliminary data confirm the utility of 2‐D electrophoretic GST analysis as indicative of lead exposure and toxicity and support its use for further elaboration of lead's effects on renal protein expression.
Incorporation of dispersion into the theory of electrophoresis of DNA and its complexes
Authors: Cann John R.
Published: May 2005

Incorporation of the dispersion coefficient into the theory of the mobility‐shift assay for DNA‐protein complexes was highly successful largely due to increased mathematical rigor. A model simulating electrophoretic migration of DNA across the phase boundary between the initial zone of macromolecule and the gel lane predicts the peak asymmetry observed experimentally. It also predicts that, under the agency of the dispersion coefficient, the peak will become progressively more symmetrical during migration along the gel lane.
Rapid detection of γT cell receptor gene rearrangements in acute lymphoblastic leukemia by electrophoresis and silver staining: Implications for detection of minimal residual disease
Authors: Valetto Angelo , Lanciotti Marina , Martino Daniela Di , Anselmi Giorgia , Bottini Federico , Mori Piergiorgio , Candiano Giovanni
Published: May 2005

Minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) was studied using polymerase chain reaction (PCR). γT cell receptor (TCRG) genes are ideal targets for PCR‐based detection of MRD due to their molecular characteristics. Polyacrylamide gel electrophoresis (PAGE) analysis of PCR products followed by silver staining was performed for 72 children with ALL at the onset of disease. Silver staining is an effective technique to detect gene rearrangements without the use of ethidium bromide. Moreover, this method may show heteroduplex bands of a clonal nature when both TCRG alleles are rearranged. PCR products subjected to a rapid staining protocol were recovered from the gel, reamplified by a second PCR and directly sequenced. After sequencing, we identified the junctional region and obtained patient‐specific probes. In more than half of the patients we detected TCRG rearrangements that were used as molecular markers for residual disease.
Characterization of DNA standards by capillary electrophoresis
Authors: Atha Donald H.
Published: May 2005

We have used capillary electrophoresis to evaluate commercial DNA size standards and have found that it can provide an efficient assessment of size. However, the accuracy of the determination is adversely affected by anomalous migration times due to specific interactions of the DNA with the gel matrix as well as conformational differences in the DNA due to sequence heterogeneity. These anomalous migration times are strongly dependent on the choice of gel matrix. For example, the anomalous migration times that are observed in a 1 kilobase standard DNA ladder can be minimized using nongel hydroxyethylcellulose. In addition, the peak resolution can be increased and the anomalous migration can be reduced by the addition of the intercalating dye, ethidium bromide. However, in the case of the D1S80 allelic ladder, some of the DNA fragments possess nucleotide sequences which do not interact equivalently with the dye and produce irregular migration times. These measurements yield preliminary information useful in evaluating DNA size standards which may be used for a wide range of DNA diagnostic applications.
Apparent pore size of polyacrylamide gels: Comparison of gels cast and run in Tris‐acetate‐EDTA and Tris‐borate‐EDTA buffers
Authors: Stellwagen Nancy C.
Published: May 2005

The electrophoretic mobilities of DNA molecules in three different molecular weight ladders were measured in polyacrylamide gels containing different acrylamide concentrations (%T) and cross‐linker ratios (%C), cast and run in Trisacetate‐EDTA (TAE) buffer. The apparent pore radius of each gel was estimated from Ferguson plots of the relative mobiliteis of each of the DNA molecules, using the mobility of the monomer fragment in each molecular weight ladder as the reference mobility. The effective size of each of the DNA molecules was estimated from its radius of gyration. The apparent gel pore radii calculated in this manner ranged from 21 nm in gels containing 10.5 %T, 5%C to 200 nm in gels with 4.6%T, 2%C; similar to the values observed for polyacrylamide gels cast and run in Tris‐borate‐EDTA (TBE) buffer (Holmes and Stellwagen, Electrophoresis 1991, 12, 612–619). Hence, the effective pore size of polyacrylamide gels is essentially independent of whether the gels are cast and run in TAE or TBE buffer.
Isolation of ultrapure supercoiled plasmid‐DNA using preparative electrophoresis
Authors: Roeder Thomas , Görich Derk , Roeder Niklas P. , Bruchhaus Iris
Published: May 2005

A large‐scale preparative polyacrylamide gel electrophoresis (PAGE) system for the isolation of high‐purity supercoiled plasmid‐DNA is described. This method should prove suitable for the isolation of large DNA molecules, either plasmid or linear DNA, that is required for the production of transgenic animals, for instance. The efficiency of the method is illustrated by the isolation of the gene for the green fluorescent protein, cloned into a mammalian expression vector and used for transfection of eukaryotic cells.
Recent developments towards a unified theory for separation science (Minireview)
Authors: Bowser Michael T. , Chen David D. Y.
Published: May 2005

Various techniques for chemical separation can be described using a generally applicable theory. There are several schools of thought on how a unified separation science should be developed. The theories described include the mass balance equation (i.e. moving boundary), virtual migration distances, and the use of individual capacity factors.