bluuebunny/crossref_metadata_2025 huggingface dataset:
The characterization of composite agarose/hydroxyethylcellulose matrices for the separation of DNA fragments using capillary electrophoresis
Authors: Siles Barbara A. , Anderson David E. , Buchanan Nathan S. , Warder Matthew F.
Published: May 2005

Mixtures of the polysaccharide derivatives, 19% hydroxyethylated SeaPrep agarose (SP‐AG) and hydroxyethylcellulose (HEC), in aqueous buffer solutions are applied for the first time to the separation of DNA fragments using capillary electrophoresis (CE). These matrices form unique size‐sieving networks that allow the separation of a wide size range of DNA fragments in a single analysis. Relative to their homogeneous counterparts, the composite separation matrices provide enhanced selectivity properties of DNA fragments, especially for fragments greater than 1000 base pairs (bp) in length. Additionally, the effects on separation performance of capillary temperature, the incorporation of a DNA intercalator, and applied field strength are demonstrated. Solution viscosity measurements of the homogeneous and composite matrix solutions were made in order to establish the entanglement threshold concentrations for the unique size‐sieving solutions. The relatively low solution viscosities of the composite separation matrices allow reproducible replacement of the separation matrix between analyses. The mechanism of separation of DNA fragments for the composite matrices is proposed.
Micropreparative capillary electrophoresis of DNA by direct transfer onto a membrane
Authors: Magnúsdóttir Soffía , Heller Christoph , Sergot Phillipe , Viovy Jean Louis
Published: May 2005

We have developed a new technique for the collection of DNA fragments separated by capillary electrophoresis, by direct transfer from the capillary outlet to a positively charged membrane. Transfer and post‐run detection of two different nonradioactivety labeled DNA standards, ranging in size from 150 bp to 2 kbp and 120 bp to 23 kbp are presented, and discussed. Capillary electrophoresis with direct blotting presents several advantages over the blotting from gels: the separation is faster and requires less manual steps, the resolution is higher, and each DNA fragment is collected into a very concentrated spot on the membrane due to the small surface of the capillary outlet and to a design of the collection device inducing a refocusing of field lines across the hybridization membrane. Therefore, very small amounts of DNA (in the pg range) can be detected. This fraction collection makes further analysis of the sample possible, e.g. by hybridization, thus suppressing one of the major present limitations of the capillary electrophoresis technique for DNA analysis

In part presented at the international meeting Electrophoresis '95, Paris, France, August 30–September 2, 1995

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Some properties of a measure of resolution in gel electrophoresis and capillary zone electrophoresis
Authors: Weiss George H. , Kiefer James E.
Published: May 2005

The most commonly used measure of resolution for chromatographic and electrophoretic separations does not take into account the possibility of there being different amounts of each of the molecular species. A modification of a measure of resolution recently suggested by Aldroubi and Garner (BioTechniques 1992, 13, 620–624) can incorporate this effect explicitly. Their criterion for resolution is based on the time to observe a valley of specified magnitude separating two peaks. We examine how this measure depends on different physically relevant parameters that characterize the system.
Generation of peptide maps by capillary zone electrophoresis in isoelectric iminodiacetic acid
Authors: Bossi Alessandra , Righetti Pier Giorgio
Published: May 2005

Capillary zone electrophoresis in stationary, isoelectric buffers is a novel method for generating peptide maps of protein digests. The buffer system developed is composed of iminodiacetic acid (IDA), whose physico‐chemical parameters were found – by theoretically modeling and experimental verification – to be: pI 2.23 (at 100 mM concentration), pK1 = 1.73 and pK2 = 2.73 (no attempts were made at measuring the pK of the primary amino group, since such a low pI value would be compatible with any pK value of the basic group, down to as low as pK 5.5). IDA is compatiable with most hydro‐organic solvents, including trifluoreoethanol (TFE), up to at least 40% v/v, typically used for modulating peptide mobility. In naked capillaries, a buffer comprising 50 mM IDA, 10% TFE and 0.5% hydroxyethylcellulose (HEC) allows generation of peptide maps with high resolution, reduced transit times and no interaction of even large peptides with the wall. However, the best background electrolyte was found to be a solution of 50 mM IDA in 0.5% HEC and 6–8 M urea, one of the best solubilizers of proteins and peptides known. In this last electrolyte system, peptide maps of β‐casein digests (known to contain also very large peptides, up to 6000 Da) could be generated with excellent resolution and half the transit times as compared with the standard buffer adopted in peptide analysis (80 mM phosphate buffer, pH 2.0). IDA thus appears to be another valid isoelectric buffer system, operating in a different pH window (pH 2.33 in 50 mM IDA) as compared to the other amphotere previously adopted (50 mM Asp, pH 2.77) for the same kind of analysis.
Detection of underivatized carbohydrates in capillary electrophoresis with a bienzyme electrode
Authors: Wei Hongping , Wang Tianlin , Li Sam F. Y.
Published: May 2005

A new approach to detect underivatized carbohydrates in capillary zone electrophoresis (CZE) with a bienzyme electrode is described. The bienzyme electrode is based on immobilization of two enzymes, glucose oxidase and amyloglucosidase, on a platinum microelectrode. Maltose and glucose were used as substrates to establish the method and study its feasibility. Using this electrode, underivatized glucose and maltose were determined after separation in 60 mM borate buffer (pH 9.3) by CZE. A liquid junction, between the surface of the bienzyme electrode and the outlet of the separation capillary, was used to lower the pH of the eluting separation borate buffer so that the immobilized enzymes could maintain high activities. The detection limits were 0.17 mM for maltose and 0.35 mM for glucose. The effects of borate ion and pH on the responses of the bienzyme electrode to maltose and glucose are discussed. The bienzyme electrode did not show any decay in responses to maltose or glucose during one week of continuous operation.
Determination of the association constant for slow phosphoserine‐anti‐phosphoserine interaction kinetics by capillary zone electrophoresis
Authors: Lin Shiming , Hsu Su‐Ming
Published: May 2005

Capillary zone electrophoresis (CZE) was used to study the interaction of a monoclonal anti‐phosphoserine antibody with its antigen. A model system that allows the determination of the real binding constant in a solution based on the change in peak areas at different concentrations of phosphoserine has been used for studying slow monoclonal antibody‐antigen interaction kinetics involving low‐molecular‐weight antigens. CZE was applied in preincubation experiments. The slow interaction kinetics led to band broadening and resulted in far lower efficiency of the separation of complexed antibody from unbound antibody. However, when the run‐to‐run reproducibility of free phosphoserine was examined, it was found that it can be recovered quantitatively under electrophoresis conditions. On the basis of measurement of peak areas at different phosphoserine concentrations, the association constant was estimated (Ka = 5.21 × 105 M−1) and shown to be in close agreement with that obtained by equilibrium dialysis (Ka = 4.65 × 105 M−1). As long as the antigen participating in the interaction can be detected and recovered quantiatively in the CZE system, the method is generally useful for the study of monoclonal antibody‐antigen interaction where the kinetics is slow and where the charge/mass ratio of the unbound antigen differs from that of the complexed molecule.
Detection and semiquantitation of albumin forms in fresh human plasma separated on gradient polyacrylamide gel by means of electroblotting on agarose gel matrix
Authors: Atmeh Ragheb F. , Shabsoug Barakat
Published: May 2005

Albumin in vitro contains several molecular forms, while in vivo it exists mainly as a monomer with a small fraction of a dimer. The aim of the present work was to detect and estimate albumin forms in fresh blood samples. The available analytical methods at present are inadequate for this purpose. An improved immunoblotting method was used where plasma was subjected to electroporetic separation on 4–25% gradient polyacrylamide gels followed by immunoblotting on agarose gel containing anti‐human albumin. The interference from the huge amount of the monomer in plasma was overcome by cutting the monomer region from the polyacrylamide gel before immunoblotting. After staining of the agarose gel, it revealed the presence of seven stained bands of albumin in addition to the monomer. These bands represent albumin aggregates and complexes of varying molecular masses (112–428 kDa). These albumin forms accounted for 0.7% of the total plasma albumin and their estimated level was 30.7 mg/dL. This study shows that the native albmin in blood has several molecular forms. It is concluded that albumin in healthy human subjects may form association complexes of varying molecular masses with other macromolecules in blood and these complexes are expected to be of physiological relevance.
Peak broadening in capillary zone electrophoresis
Authors: Gaš Bohuslav , Štědrý Milan , Kenndler Ernst
Published: May 2005

A review on peak broadening in capillary zone electrophoresis in free solutions is given which covers a selection of the literature published on this topic over the period mainly between 1992 and the beginning of 1997 (consisting of 71 publications). The contributions to peak dispersion from extracolumn effects (e.g. due to the finite length of the injection zone, or the aperture of the detector), from longitudinal diffusion, Joule heating, electromigration dispersion (concentration overload), a different path length of the solute ions, wall adsorption, laminar flow and the (longitudinally) homogeneous or nonhomogeneous electroosmotic flow are described. The latter may also occur when a longitudinally nonhomogeneous radial electric field is applied. Peak dispersion is depicted either by the plate‐height model, or the concentration of the solute as a function of space and time is calculated either analytically or numerically by solving the equation of continuity with appropriate initial and boundary conditions and possibly completed by equations governing further quantities.
Recent application and developments of capillary isotachophoresis
Authors: Gebauer Petr , Boček Petr
Published: May 2005

Capillary isotachophoresis is a powerful electromigration separation method with a pronounced capability to concentrate trace components in diluted samples. At present, capillary isotachophoresis is utilized predominantly as the first step in on‐line combination with capillary zone electrophoresis. This article is a continuation of previous reviews and summarizes the results published during 1993–1996.
Advances in capillary electrochromatography
Authors: Colón Luis A. , Reynolds Kimberly J. , Alicea‐Maldonado Rafael , Fermier Adam M.
Published: May 2005

Capillary electrochromatography (CEC) is a hybrid between capillary electrophoresis and high performance liquid chromatography (HPLC) that has gained popularity in recent years. CEC uses an electrically driven flow to transport the solutes through the chromatographic column. Separation can be achieved by differential partition between two phases, differential electromigration, or a combination of these two. Herein, the main features of CEC are presented, including basic principles and a literature overview on different practical approaches used.
Capillary electrophoresis in presence of sodium dodecyl sulfate and a sieving medium
Authors: Takagi Toshio
Published: May 2005

For analysis of proteins in presence of sodium dodecyl sulfate (SDS), basic knowledge about their behavior in presence and absence of sieving medium is required. Capillary electrophoresis (CE) is particularly suitable for this purpose. Although various polymers are used as sieves in CE analysis in presence of SDS, attention must be paid to possible interaction between SDS and the polymer. For obtaining new insights polymers with defined molecular weight and distribution should be utilized.
Recent advances in capillary electrophoresis/electrospray/mass spectrometry
Authors: Banks J. Fred
Published: May 2005

Successful on‐line interfacing of capillary electrophoresis (CE) with electrospray (ES) mass spectrometry (MS) has progressed substantially in recent years. Of particular note also is the development which has occurred in combining the more advanced capillary‐based electromigration separation techniques, such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CIT), micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC), with ES/MS. The union of these electromigration schemes with MS detection provides a useful and sensitive analytical tool for the separation, quantitation and identification of biological, therapeutic, environmental and other important classes of chemical analytes. By making optimal use of the characteristics inherent with these separation mechanisms, greatly enhanced MS performance may be obtained. The following review summarizes the significant issues and challenges involved with CE/ES/MS analysis as well as results which have recently been obtained.
Electrochemical detection in capillary electrophoresis
Authors: Voegel Phillip D. , Baldwin Richard P.
Published: May 2005

Recent advances in the design and application of electrochemical detection (EC) systems in capillary electrophoresis (CE) are reviewed, with the objective of providing the non‐electrochemist with a state‐of‐the‐art picture of CEEC instrumentation and an overview of the primary analytes and samples for which the technique is best suited. In particular, instrument innovations designed to aid in decoupling the CE and EC systems electrically and in aligning them physically are described in detail. In addition, CEEC applications are summarized for four specific analyte groups: catecholamines, thiols and disulfides, amino acids, and carbohydrates. On this basis, it is clear that EC techniques have reached a stage where they are already having a significant impact on CE usage in selected areas of analysis. Continued developments with respect to new electrode materials and electrode configurations promise to broaden this impact further.
Critical review of recent developments in fluorescence detection for capillary electrophoresis
Authors: MacTaylor Christine E. , Ewing Andrew G.
Published: May 2005

Recent developments in capillary electrophoresis with fluorescence detection are reviewed. Instrumental advances have led to increased sensitivity and, therefore, a growing number of applications. Capillary electrophoresis has been coupled with various techniques to achieve multi‐dimensional separations. Other advances have focused on temporal resolution when sampling from biological environments, increased sample throughput especially for DNA analysis, and fast separation times. New technologies including chip and channel electrophoretic separations with fluorescence detection are also discussed.
Chiral separations in capillary electrophoresis using proteins as stereoselective binding agents
Authors: Hage David S.
Published: May 2005

Proteins are receiving increased attention in capillary electrophoresis (CE) for use as ligands in chiral separations. This review examines the types of proteins that have been employed in CE for the analysis of chiral compounds and provides a summary of applications for such an approach. Several formats for this type of analysis are discussed, including methods that use immobilized proteins (i.e., affinity capillary electrochromatography and capillary gel affinity electrophoresis) or techniques that use proteins as running buffer additives (i.e., affinity capillary electrophoresis). The role of various experimental factors in these separations are considered, and a comparison is made between the general analytical properties of CE systems that use immobilized versus solution‐phase proteins.
Chiral surfactants in micellar electrokinetic capillary chromatography
Authors: Camilleri Patrick
Published: May 2005

Most biologically active molecules contain one or more chiral centres, giving rise to stereoisomeric forms which can behave differently in a chiral environment. Thus, only one of the enantiomers may show the desired physiological activity, whereas the other enantiomers may either be considerably less active or even show undesireable side effects. Establishing that a chiral drug consists of one single enantiomer is nowadays essential before it can be given to patients. Analytical tools that can discriminate between enantiomers play a very important role in determining the stereoisomeric composition of chiral molecules. Until recently chromatographic techniques were the most popular for enantiomeric separations. The increased use of capillary electrophoresis (CE) has provided complementary methodology for chiral discrimination. One mode of CE that has been used for this purpose is micellar electrokinetic capillary chromatography (MECC), where natural or synthetic chiral surfactants are added to the separation buffer.
Behavior of peptides in capillary electrophoresis: Effect of peptide charge, mass and structure
Authors: Cifuentes Alejandro , Poppe Hans
Published: May 2005

In the last decade the large potential of capillary electrophoresis as a technique for separation and characterization of peptides has been demonstrated extensively. In this field, a large number of chemical structures has to be taken into consideration, for which very often no data or even standards are available. As a result, there has been a strong desire to relate electrophoretic behavior to molecular properties and structure of the compounds. The activities in that direction, in the area of capillary zone electrophoresis, are critically reviewed. Special attention is paid to peptide charge, mass, hydrophobicity and structure, and their influence on the selectivity of the separation. Also, some complexation phenomena are discussed.
Recent developments in capillary electrophoresis of carbohydrate species
Authors: El Rassi Ziad
Published: May 2005

Carbohydrates are ubiquitous species involved in many life processes. Because of the multilateral roles of carbohydrates, their analysis has come to have increasing importance. As shown in this review, capillary electrophoresis in its various modes of operation has proved very useful in the analysis of carbohydrate species including mono‐ and oligosaccharides, glycoproteins, glycopeptides and glycosaminoglycans. Advances in separation approaches and applications as well as advances in detection including sensitive and selective precolumn derivatization are described. In summary, this comprehensive review is a supplement to previous reviews and covers the published work in 1996 and the first half of 1997.
Determination of metal ions by capillary electrophoresis
Authors: Macka Miroslav , Haddad Paul R.
Published: May 2005

The increasing use of capillary electrophoresis (CE) in all areas of analytical chemistry has been reflected in the appearance since 1990 of significant numbers of fundamental studies and applications of CE in the area of separations of metal ions. In this article all aspects of separations of metal ions by CE are reviewed and discussed, including general concepts and approaches, sample introduction, complex formation and sample stacking, separation selectivity, the role of kinetics, capillary wall chemistry, detection techniques, method validation, and applications to metal determinations in various matrices.
Membrane transport in rat liver endocytic pathways: Preparation, biochemical properties and functional roles of hepatic endosomes
Authors: Pol Albert , Enrich Carlos
Published: May 2005

The endocytic compartment has emerged as a major regulator of the uptake and processing of circulating ligands, and has been extensively studied during the last decade. In this work, the polypeptides of the three endosomal fractions: compartment of uncoupling receptors and ligands (CURL), multivesicular bodies (MVB) and receptor recycling compartment (RRC), isolated from livers of estradiol‐treated rats, were analyzed by two‐dimensional gel electrophoresis. Silver‐stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approximately 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated. The polypeptide composition of the bile was also studied and compared with ligands and proteins identified in the different endosomal fractions. One‐ and two‐dimensional gel electrophoresis and Western blotting were used to investigate the protein composition of the three isolated endocytic fractions and 39 proteins were identified. The distribution of identified receptors, ligands and structural proteins among the three endosomal fractions was in agreement with their expected functionalities and with the different endocytic pathways in the hepatocyte.
Two‐dimensional gel electrophoresis analysis of endovacuolar organelles
Authors: Scianimanico Sandra , Pasquali Christian , Lavoie Jacques , Huber Lukas A. , Gorvel Jean‐Pierre , Desjardins Michel
Published: May 2005

Cells perform their multiple functions with the aid of a series of distinct membrane organelles. In the last years, many of these compartments have been isolated, purified, and extensively studied. The major roles of each organelle in the cell are well understood. However, most of the molecular basis by which they perform their functions is poorly known. The recent identification and study of a handful of proteins associated with endovacuolar compartments has had a major impact on the understanding of the molecular details of organelle functions even though two‐dimensional (2‐D) gel analysis indicates that hundreds of proteins are typically associated with a complex organelle. This shows that many details and surprises are still to come for cell biologists. In the present study, we have analyzed and compared different organelles of the endocytic and phagocytic apparatus using 2‐D gel electrophoresis.
Generation of proteoliposomes from subcellular fractions
Authors: Rojo Manuel , Budin Nicolas , Kellner Roland , Gruenberg Jean
Published: May 2005

Intracellular membranes are highly dynamic, yet they retain their identity and functional characteristics. Integral membrane proteins, which must confer this specific membrane identity, remain poorly characterized at the biochemical level, largely because detergent‐mediated solubilization is required for purification and analysis, and several properties of integral membrane proteins can only be investigated when the molecule is properly embedded in a lipid bilayer. We present a method for the efficient reconstitution into proteoliposomes of integral membrane proteins from subcellular fractions. Integral membrane proteins were identified on high‐resolution two‐dimensional gels after selective extraction of soluble and peripheral membrane proteins; they accounted for 8% of the number of resolved polypeptides. A reconstitution procedure based on membrane solubilization with dodecyl‐octaoxyethylene (C12E8) and subsequent detergent removal with BioBeads SM‐2 resulted in the efficient reconstitution of several membrane proteins into proteoliposomes of uniform density. The generated proteoliposomes strongly resemble the starting membrane fraction in protein composition. This reconstitution allows the functional characterization of integral membrane proteins after enrichment and/or specific (immuno)depletion.
A novel epitope tag for the detection of rabGTPases
Authors: Nagelkerken Bas , Mohrmann Karin , Gerez Lisya , van Raak Marcel , Leijendekker Richtje , van der Sluijs Peter
Published: May 2005

Rab GTPases are localized on the cytoplasmic surface of most intracellular organelles where they play a role in the regulation of vesicular transport. As it has been difficult to detect endogenous rab proteins by morphological methods, their localizations were often inferred from transfection experiments using epitope‐tagged constructs. Because most of the available epitope tags are only recognitzed by mouse monoclonal antibodies they are often not suitable for double or triple label immunocytochemistry. To overcome this problem, we generated antibodies against a novel 10 amino acid X31 influenza hemagglutin epitope (NH). We here characterized these antibodies and document their utility for detecting early endosomal rab proteins N‐terminally tagged with the NH decapeptide in morphological and biochemical assays.
Make2ddb: A simple package to set up a two‐dimensional electrophoresis database for the World Wide Web
Authors: Hoogland Christine , Baujard Vincent , Sanchez Jean‐Charles , Hochstrasser Denis F. , Appel Ron D.
Published: May 2005

Two‐dimensional electrophoresis (2‐DE) has become a highly reproducible protein separation technique that currently serves as the main basis for proteome research and in particular for protein identification. Also, the Internet provides large utilities for exchanging data, and we can observe increased interest among scientists to build remote 2‐DE databases, since many members of the concerned community are now able to access the data. By preparing the data and programs that are required to create a federated 2‐DE database, the Make2ddb package, described here, helps to build such a database on the user own World Wide Web site.
Rapid access to biomedical knowledge with GeneCards and HotMolecBase: Implications for the electrophoretic analysis of large sets of gene products
Authors: Rebhan Michael , Prilusky Jaime
Published: May 2005

Rapid access to well‐organized information about gene products is important for many studies that simulatenously monitor large sets of those factors, for example with electrophoretic methods. HotMolecBase and GeneCards, Internet resources that may be accessed from our Bioinformatics homepage at http://bioinfo.weizmann.ac.il/, have been designed to address similar problems. GeneCards presents semi‐automatically collected information about all approved human genes and their products (with a focus on cellular functions and medical aspects), and offers a new kind of knowledge navigation guidance system that interactively guides the information‐seeking scientist to relevant information. On the other hand, HotMolecBase is a collection of more extensive hypertext fact sheets about a small set of medically interesting molecules (mainly proteins) that are regarded as especially promising targets for drug development. Together, both resources may help scientists world‐wide to find their way in the growing labyrinth of biomedical information on the World Wide Web. In the present article, we want to explain how these resources may be used by researchers who want to access information related to particular spots on two‐dimensional electrophoresis gels.
Establishment of the human reflex tear two‐dimensional polyacrylamide gel electrophoresis reference map: New proteins of potential diagnostic value
Authors: Molloy Mark P. , Bolis Shirley , Herbert Ben R. , Ou Keli , Tyler Margaret I. , van Dyk Derek D. , Willcox Mark D. P. , Gooley Andrew A. , Williams Keith L. , Morris Carol A. , Walsh Bradley J.
Published: May 2005

To understand the changes in protein expression associated with various physiological states as well as the development of pathological eye disease, we have begun to map the protein components of normal human reflex tears. An analytical reference map of normal human reflex tears was created using two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) with pH 3.5–10 immobilized pH gradients (IPGs). Micropreparatively loaded gels were transferred to polyvinylidene difluoride (PVDF) and analysed by a combination of N‐terminal sequence tagging and amino acid compositional analysis. Thirty spots were sequence tagged, resulting in identification of six different proteins (lipocalin, lysozyme, lactotransferrin, zinc‐α‐2 glycoprotein, cystatin S, cystatin SN) that matched to entries in the SWISS‐PROT database. A group of N‐terminally blocked proteins was clearly identified from SWISS‐PROT by amino acid analysis, isoelectric point (pI) and molecular weight (Mr). A number of highly expressed protein components remain unidentified despite being subjected to amino acid analysis and Edman sequencing. A majority of the abundant proteins showed varying degrees of charge heterogeneity attributed to post‐translational processing such as glycosylation and N‐terminal truncation. We have identified a previously undescribed protein that we have named lacryglobin. This protein displays strong homology with mammaglobin, a protein overexpressed in breast cancer. The discovery of this homologue in tears offers the potential for disease diagnosis by screening tear fluid proteins.
Standardized characterization of gene expression in human colorectal epithelium by two‐dimensional electrophoresis
Authors: Reymond Marc A. , Sanchez Jean‐Charles , Hughes Graham J. , Günther Klaus , Riese Jutta , Tortola Silvia , Peinado Miguel A. , Kirchner Thomas , Hohenberger Werner , Hochstrasser Denis F. , Köckerling Ferdinand
Published: May 2005

New diagnostic and prognostic markers are needed in colorectal cancer. They can be found by differential analysis at DNA, RNA or protein level. The accuracy of phenotypic comparisons of tumor and normal tissues depends on the purity of the samples. We present an effective method to identify and isolate proteins that are differentially expressed under altered conditions, and a two‐dimensional reference protein map of the normal human colonic epithelium Normal colonic mucosa, primary tumors and liver metastases were prepared in the operating room. After washing in an ice‐cold medium containing protease inhibitors, crypts were isolated by mechanical preparation without using metalloproteinases. Epithelial cells were then selected using Ber‐EP4 Dynabeads. The samples were denaturated before processing for immobilized pH gradient two‐dimensional polyacrylamide gel electrophoresis according to SWISS‐2DPAGE standards. The samples contained more than 95% epithelial cells as confirmed by fluorescence‐activated cell sorting using pan‐anticytokeratin antibodies. Cell surfaces were not damaged, as assessed by scanning electronic microscope. A protein reference map of the normal colonic epithelium was defined. Using gel matching, N‐terminal sequencing and/or immunoblotting techniques, 60 polypeptides – including proteins specifically expressed in colorectal epithelium – have now been identified. This reproducible method of sample preparation permits the comparison of protein patterns found in various pathological states with the present reference map (http://www.expasy.ch). Some of these patterns might provide diagnostic or prognostic markers, or even molecular targets for therapy in the future.
Mitochondrial D‐loop 3′ (CA)n repeat polymorphism: Optimization of analysis and population data
Authors: Szibor Reinhard , Michael Matthias , Spitsyn Victor A. , Plate Ines , Ginter Evgeni K. , Krause Dieter
Published: May 2005

We report a dinucleotide repeat polymorphism in the 3′ area of the mitochondrial control region. The fragments obtained using a new primer set could be reliably separated by polyacrylamide gel electrophoresis (PAGE) using nondenaturing gels. A total of five alleles [(CA)3 to (CA)7] were detected on silver‐stained gels. The 90 bp product corresponds to allele 5. Samples from one African and three European populations were characterized. Significant differences could be demonstrated as to the incidence of single alleles and allele distributions in different populations. These differences were found between the three European and one African Bantu population. For specific forensic questions the mitochondrial CA repeat is well suited. Gene diversities in populations of Germany, Hungary, the Russian Federation and Cameroon were 0.36, 0.40, 0.34, 0.52, respectively.
Electrophoresis of DNA sequencing fragments at elevated temperature in capillaries filled with poly(N‐acryloylaminopropanol) gels
Authors: Lindberg Peter , Righetti Pier Giorgio , Gelfi Cecilia , Roeraade Johan
Published: May 2005

The performance of poly(N‐acryloylaminopropanol) (poly AAP) gel columns, proved to be stable during electrophoresis at elevated temperature, was investigated. The column manufacturing procedure included the preparation of a coating of the inner wall of the fused silica capillary column with linear poly(AAP). Then, a mixture of the AAP monomer, the cross‐linker dihydroxyethylenebisacrylamide (DHEBA) and linear poly(AAP) was introduced into the column and in situ polymerized (for preparation of linear gel columns, the addition of DHEBA was omitted). The poly(AAP) columns were first evaluated by electrophoresis of oligonucleotides at room temperature and at 50°C, utilizing 260 nm UV‐absorbance detection. In a further evaluation of column performance, samples of T‐terminated DNA Sanger fragments from the bacteria Moraxella were separated at 200 V/cm electrical field strength, utilizing a 488 nm argon ion laser and a confocal optical setup for laser‐induced fluorescence (LIF) detection. A temperature increase from 25°C to 50°C effectively released a compression of DNA bands. However, for cross‐linked poly(AAP) gel columns, the elevated temperature resulted in a considerable reduction of the DNA sequence reading length. When a linear poly(AAP) column was utilized, no detrimental effect of elevated temperature on the separation could be observed.
Application of capillary electrophoresis for the determination of inorganic ions in trace explosives and explosive residues
Authors: Kishi Tohru , Nakamura Jun , Arai Hiroyuki
Published: May 2005

Capillary electrophoresis was developed for the analysis of low explosive residue, because a significant amount of inorganic anions and cations remain after deflagration. Certain high explosives, such as emulsion explosives, produce a vast quantity of inorganic ions after a blast and can readily be analyzed using capillary electrophoresis. Often, trace amounts of explosive residues may be present on physical evidence submitted in criminal cases. Trace amounts of inorganic ions such as nitrate, chlorate, and ammonium may be detected using capillary electrophoresis owing to the low detection limit of these species. The utility of capillary electrophoresis in the analysis of explosive residues is in its ability to simultaneously analyze trace explosives and ionic products present on physical evidence.
Capillary electrophoretic determination of trace metals in hair samples and its comparison with high performance liquid chromatography and atomic absorption spectrometry techniques
Authors: McClean Stephen , O'Kane Edmund , Coulter Daniel J. M. , McLean Stephen , Smyth W. Franklin
Published: May 2005

Capillary electrophoresis (CE) is compared with high performance liquid chromatography (HPLC) and atomic absorption spectrometry (AA) for the determination of trace concentrations of selected metals in human hair. CE and HPLC methods are based upon the chelation of the metals with 2‐(5′‐bromo 2′‐pyridylazo)‐5‐diethylamino phenol (5‐Br‐PADAP) followed by ultraviolet‐visible (UV‐Vis) detection. Large volume sample stacking (LVSS)‐CE using injection times of up to 300 s is applied to the simultaneous determination of Co and Zn providing lower limits of detection (LODs) of 4.2 × 10−8 mol dm−3 and 6.0 × 10−8 mol dm−3, respectively, than can be achieved by conventional CE. The LVSS procedure could not be applied to hair samples due to a higher run current existing when the sample is introduced into the capillary. Conventional CE with cetyltrimethylammonium bromide (CTAB) present in the run buffer to retain the ligand in solution is used for the determination of metal concentrations in hair samples. Only Zn could be determined in this way as it exists at relatively high levels in hair. Co, Ni and Hg could be detected when spiked hair samples were analysed with estimated LODs of 5.0 × 10−7 mol dm−3, 1.0 × 10−6 dm−3 and 3.0 × 10−5 mol dm−3, respectively. HPLC was successfully used to determine Co and Cu in hair samples, with levels of 57.6 ppb and 17.31 ppm being found, respectively, and corresponded closely to results produced by AA. Fe also gave a signal together with unidentified coeluting species. In a separate HPLC study the determination of Ni and Hg as their complexes with hydrogen peroxide and 5‐Br‐PADAP was also investigated and LODs of 2.0 × 10−6 mol dm−3 and 5.0 × 10−6 mol dm−3, respectively, were achieved. AA was used as a reference method to determine levels of Co, Cu, Fe, Pb and Zn in hair and the results produced were in order of magnitude agreement with accepted literature values.
Application of high‐performance capillary electrophoresis to the quantitative analysis of nicotine and profiling of other alkaloids in ATF‐regulated tobacco products
Authors: Lu Giang Hoang , Ralapati Suresh
Published: May 2005

Tobacco products regulated by the Bureau of Alcohol, Tobacco and Firearms (ATF), are classified at different excise tax rates according to the Code of Federal Regulations. These include the smoking (cigars, cigarettes, pipe tobacco and roll‐your‐own) and smokeless (chewing tobacco and snuff) tobacco products. The active principal components in all tobacco products belong to a class of compounds known as alkaloids. Nicotine is the major tobacco alkaloid, comprising about 98% of the total alkaloids. It is also the primary determinant of what constitutes a tobacco product from a regulatory standpoint. Nornicotine, anabasine and anatabine constitute the minor tobacco alkaloids of importance and interest to ATF. We have previously shown capillary electrophoresis (CE) to be a powerful analytical tool for monitoring nicotine in ATF‐regulated products. Here we have extended those CE studies to (i) quantitate nicotine in ATF‐regulated tobacco products and (ii) to characterize these different tobacco products according to their alkaloid profiles. Results from these studies will be presented.
Can amphoteric substances with very small (or negative) ΔpK difference exist and what properties would their water solutions exhibit?
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio
Published: May 2005

For calculating the buffer capacity of ampholytes an application of the stepwise and parallel dissociation models is considered. It is demonstrated that the scheme with stepwise dissociation possesses a “nonadditive” sum, i.e., the resulting buffer capacity of the system exceeds the sum of the separate contributions of the two ionogenic groups. On the other hand, the scheme of parallel dissociation is free from this disadvantage, and thus does not impose any restriction on the ΔpK parameter.
Isotachophoresis at pH extremes: Theory and experimental validation
Authors: Ermakov Sergey V. , Zhukov Michael Y. , Capelli Laura , Righetti Pier Giorgio
Published: May 2005

The evolution of an isotachophoresis (ITP) system in acidic or basic pH ranges can be quite different from that predicted by the existing theory. It was found theoretically and proved experimentally that the contribution of hydrogen or hydroxyl ion to conductivity of solution and/or its net charge changes the behavior of the ITP system, creating in the terminating electrolyte an additional zone close to the initial interfaces between electrolytes (leader and terminator). One boundary of the zone, being either sharp or dispersed, moves toward the leader; the other is always sharp and stationary and coincides with initial electrolytes' discontinuity. The latter can be registered in the presence of electroosmotic flow which delivers it to the detection point. In order to describe the dynamics of the ITP system at pH extremes an algorithm of analytical solution was developed, based on the revised Kohlrausch theory. Its predictions coincide well with computer simulations and experimental data. The results presented can help in a correct analysis of ITP data and explain some confusing phenomena which were considered to be artifacts.
Separation of 4‐color DNA sequencing extension products in noncovalently coated capillaries using low viscosity polymer solutions
Authors: Mandabhushi Ramakrishna S.
Published: May 2005

A low viscosity (ca. 75cp) solution using polydimethylacrylamide (PDMA) was developed for separating DNA sequencing extension products by capillary electrophoresis (CE). This medium gave a length‐of‐read (LOR) value of approximately 600 bases in about 2 h using four‐color sequencing in 50 μm capillary at 42°C under a field of 160 V/cm. This medium also works in bare capillaries by noncovalently coating the surface to suppress both electroosmotic flow (EOF) and DNA‐capillary wall interactions, and eliminates the need for complicated covalent coatings. At least 100 successive sequencing runs were performed in the same capillary by simply pumping fresh medium after every run, without requiring any reconditioning of the capillary surface between runs. The thermal stability of the noncovalent coating can be improved by adding small amounts of high molecular weight PDMA to the separation medium. The advantages of low viscosity separation media and uncoated capillaries are of paramount importance to develop high‐throughput instruments for DNA sequencing.
Capillary electrophoresis of peptides and proteins in fused‐silica capillaries coated with derivatized polystyrene nanoparticles
Authors: Kleindienst Gerald , Huber Christian G. , Gjerde Douglas T. , Yengoyan Leon , Bonn Günther K.
Published: May 2005

High‐resolution capillary electrophoretic separation of proteins and peptides was achieved by coating the inner wall of 75 μm ID fused‐silica capillaries with 40‐140 nm polystyrene particles which have been derivatized with α‐ω‐diamines such as ethylenediamine or 1, 10‐diaminodecane. A stable and irreversibly adsorbed coating was obtained upon deprotonation of the capillary surface with aqueous sodium hydroxide and subsequent flushing with a suspension of the positively charged particles. At pH 3.1, the detrimental adsorption of proteins to the capillary inner wall was suppressed efficiently because of electrostatic repulsion of the positively charged proteins from the positively charged coating which enabled protein separations with maximum efficiencies of 400 000 plates per meter. A substantial improvement of separation efficiency in particle‐coated capillaries was observed after in‐column derivatization of amino functionalities with 2,3‐epoxy‐1‐propanol, resulting in a more hydrophilic coating. Five basic and four acidic proteins could be separated in less than 7 min with efficiencies up to 1 900 000 theoretical plates per meter. Finally, coated capillaries were applied to the high‐resolution analysis of protein glycoforms and bioactive peptides.
Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis (II)
Authors: Schmid Martin G. , Gübitz Gerald , Kilár Ferenc
Published: May 2005

Further studies on chiral resolution of drugs with different chemical structures by capillary zone electrophoresis using iron‐free human serum transferrin are described. The substances passed a highly concentrated pseudo‐stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Eighteen drugs with different structures were screened, and the enantiomers of clofedanol, buphenine, acebutolol and chlorphenamine were resolved. Several, but not all drugs, showed longer migration times while passing the protein zone, indicating an interaction with transferrin, although chiral resolution was not observed in all cases. The observations provided further information about the properties of the surface interaction sites of transferrin.
Quantitative trace analysis of interleukin‐3, interleukin‐6, and basic model proteins using isotachophoresis‐capillary zone electrophoresis with hydrodynamic counterflow
Authors: Bergmann Jens , Jaehde Ulrich , Schunack Walter
Published: May 2005

A quantitative analytical technique to determine trace concentrations of recombinant human interleukin‐3 (rhIL‐3), recombinant human IL‐6 (rhIL‐6), and various basic model proteins is described using isotachophoresis‐capillary zone electrophoresis (ITP‐CZE). Proteins were separated on coated fusedsilica capillaries using a commercial capillary electrophoretic system modified for the application of isotachophoretic preconcentration with hydrodynamic counterflow. The effect of injection time and isotachophoretic focusing time was investigated and compared with predictions from existing mathematical models. Good linearity of the calibration graphs (r > 0.995) was observed for all investigated proteins. The limit of quantification was in the 10−8 M range using UV detection at 200 nm. Within‐day and between‐day precison of peak area ranged between 1 and 6%. Precision was unaffected by isotachophoretic preconcentration. In conclusion, the described method is feasible to quantify trace concentrations of rhIL‐3, rhIL‐6, and basic proteins. Potential applications comprise issues of pharmaceutical quality control.
Capillary affinophoresis of pea lectin with polyliganded affinophores: A model study of divalent‐polyvalent interactions
Authors: Shimura Kiyohito , Kasai Ken‐Ichi
Published: May 2005

Affinophoresis is a type of affinity electrophoresis using an affinophore, a soluble ionic carrier bearing affinity ligand(s). It was reported previously that an affinophore, prepared by coupling multiple p‐aminophenyl α‐D‐mannoside ligands to a part of the carboxyl groups of succinylated polylysine, specifically changed the mobility of pea lectin in agarose gel. The affinophoresis of this divalent lectin with the polyliganded affinophore was investigated by using capillary electrophoresis. Analysis of the mobility change of the lectin in the presence of differently modified affinophores showed that the affinity was larger for affinophores having higher ligand density. Analysis of the inhibition of the mobility change by a neutral ligand, with a known affinity constant for the lectin, allowed estimation of the contributions of monovalent and divalent interactions to the binding in the lectin‐affinophore complex. The proportion of divalent complexes was greater for affinophores having higher ligand density. This approach to estimate the contribution of divalency in complex formation should be generally applicable to the analysis of divalent interactions with different techniques other than electrophoresis.