bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of pH‐dependent structural transition in telomeric sequences by two‐dimensional gel electrophoresis
Authors: Belotserkovskii Boris P. , Frank‐Kamenetskii Maxim D.
Published: May 2005

We have applied the method of two‐dimensional gel electrophoresis to study a pH‐dependent structural transition in plasmid carrying the Tetrahymena telomeric sequence, (G4T2)n. A study of the two‐dimensional patterns makes it possible to obtain energetic characteristics of the transition. We have obtained the nucleation energy of the structure. It proved to be two times lower than the nucleation energy of H‐DNA determined before for a d(AG)n insert. The finding partly explains the formation of an eclectic structure in telomeric sequences. We also studied the effect of Zn++ ions on the two‐dimensional patterns of gel electrophoresis. Our data show that Zn++ ions can significantly affect the transitions themselves. This finding leads to reinterpretation of numerous data on probing of structural transitions in DNA by the S1 nuclease.
DNA electrophoresis in polymer solutions: Ogston sieving, reptation and constraint release
Authors: Viovy Jean‐Louis , Duke Thomas
Published: May 2005

The electrophoresis of long polyelectrolytes is considered theoretically, with special attention to duplex DNA. We first discuss quantitative approaches to determine unambiguously the entanglement properties of polymer solutions. Following an idea proposed by Grossman and Soane, we then assume that the “mesh” size of the solution plays the role of a dynamic “pore size” in order to apply theories for gel electrophoresis. In the framework of the Ogston model, we predict that duplex DNA up to 1 kb or more should be separable in dilute (i.e. nonentangled) solutions of high molecular weight polymers. In an entangled solution, and for DNA larger than the pore size, we use a recently developed fluctuation‐reptation model to predict the range of sizes in which separation should be possible as a function of electric field E and pore size ξ b. For ξb larger than the Kuhn length of DNA, we predict a separation up to a size N*scaling as E−1ξb−1. For ξb smaller than the Kuhn length, two different regimes are expected. For small electric fields (typically of the order of 10 V/cm), N*should be proportional to E−1ξb−3/3, whereas for high electric fields such as encountered in capillary electrophoresis, we expect that N*is proportional to E−2/5ξb12. These predictions are qualitatively different from earlier ones. Finally, we demonstrate that the finite lifetime of the “pores” in anentangled solution (as opposed to a gel) may lead to a new migration mechanism by constraint release, which is not size‐dependent. In contrast with earlier suggestions, we show that, in general, the concentration should be raised above a minimal value significantly higher than the entanglement threshold c*in order to separate large DNA molecules. We propose expressions for this minimal concentration as a function of DNA and polymer sizes. This model suggests that, for a given high molecular weight polymer, the size of the largest DNA that can be separated increases roughly linearly with the viscosity of the solution.
Cellular automaton simulation of pulsed field gel electrophoresis
Authors: Smith Mark A. , Bar‐Yam Yaneer
Published: May 2005

We describe simulation techniques well suited to detailed investigation of the microscopic behavior of DNA during electrophoretic separation in the diffusive regime. Long polymers moving diffusively in a medium are simulated using microscopic Monte‐Carlo steps. Simulations rely upon a recently introduced two‐space abstract polymer that enables fine‐grained massively parallel simulation. Tests of the two‐space polymer dynamics are reviewed. The scaling with polymer length of the size and relaxation time of isolated polymers are shown to agree with universal scaling relations. The relaxation time is found to be significantly faster than the alternative bond‐fluctuation method. Simplicity of implementation enables simulation on cellular automaton machines (CAM) including CAM‐6, and a prototype of the new CAM‐8, as well as other massively parallel architectures. Preliminary simulations of polymers migrating under an external field through a random medium of obstacles in two dimensions are described. Two sequences of simulations are performed, with different obstacle densities corresponding to pore sizes larger and smaller than the polymer radius of gyration. In the dilute medium polymers are characteristically draped on single obstacles. In the denser medium draping across multiple obstacles results in reduced orientation in the field direction. A demonstration of rapid 90° field direction switching results in polymer motion toward the expected intermediate direction.
A new type of pH‐ and detergent‐stable coating for elimination of electroendosmosis and adsorption in (capillary) electrophoresis
Authors: Hjertén Stellan , Kubo Kanenobu
Published: May 2005

We describe a method to coat fused silica, quartz, or glass electrophoresis tubes with hydrophilic polymers, such as methyl cellulose or dextran, in order to eliminate electroendosmosis and adsorption of solutes onto the tube wall. The coating was not split off upon exposure to a 5% solution of sodium dodecyl sulfate (SDS) at pH around 12 during a period of sixteen days or to 0.5 M HCI (pH 0.3) during 10 days. The high stability of the coating, which is attached to the silica surfaceviaSi‐O‐Si‐C bonds, permits electrophoresis experiments at extreme (besides neutral) pH values and allows short washes of the electrophoresis tubes between the runs with 0.05 M sodium hydroxide (pH 12.7) containing 5% SDS, and with 1 M HCI. These washing solutions release efficiently those solutes which have a tendency to adsorb onto the coating, which is a prerequisite for reproducible runs. The quality of the coating was strongly affected by the surface structure of the commercial fused silica tubing, which may vary from batch to batch. Attempts to standardize the surface structure of any tubing by treatments with NaOH and HCI to obtain a coating, the quality of which is independent of the original surface structure, were not successful. Therefore, it may be necessary to modify certain details of the coating procedure for each new batch of tubing to obtain optimum stability. Alternative coating procedures are therefore described, one of which is of general applicability (see last paragraph in Section 4).
Studies in capillary zone electrophoresis with a postcolumn multiple capillary device for fraction collection and stepwise increase in electroosmotic flow during analysis
Authors: Nashabeh Wassim , Smith Joel T. , Rassi Ziad El
Published: May 2005

A new approach involving the stepwise increase in electroosmotic flow during analysis in capillary zone electrophoresis is introduced and evaluated in the rapid separations of proteins and peptides. The stepwise increase in electroosmotic flow is based on the principle of coupled capillaries in series having different flow characteristics, a concept that was introduced recently by our laboratory. To produce stepwise changes in electroosmotic flow during analysis, a post‐column multiple capillary device, which allowed the switching between several coupled capillary systems, was designed and constructed in‐house. The utility of the multiple capillary device was also demonstrated and extended to fraction collection of separated analytes in short capillary segments. The fraction collection in capillaries facilitated the quantitative transfer of the collected fractions to high performance liquid chromatography (HPLC) for further analysis or to mass spectrometry (MS) for structural determination. The off‐line combination of capillary zone electrophoresis with HPLC or MS utilized commercial instruments without the need of expensive interfacing designs.
Use of small‐diameter capillaries for increasing peptide and protein detection sensitivity in capillary electrophoresis‐mass spectrometry
Authors: Wahl Jon H. , Goodlett David R. , Udseth Harold R. , Smith Richard D.
Published: May 2005

The use of small ID capillaries is shown to provide a substantial increase in sensitivity for capillary electrophoresis‐electrospray ionization/mass spectrometry (CE‐ESI/MS). In a comparison using capillaries ranging from either 100 to 10 μm or 50 to 5 μm ID and chemically modified with aminopropylsilane, a 25‐ to 50‐fold increase in sensitivity was observed for both peptide and protein mixtures. This enhanced solute sensitivity allowed the detection of approximately 150 attomoles of melittin (2845 Da) with selected ion monitoring and 600 attomoles of carbonic anhydrase (29157 Da) while scanning for CE‐MS with a quadrupole mass spectrometer. For the protein mixture, mass spectra of sufficient quality for precise molecular weight determination (⩽ 0.05%) were obtained for 600 attomole injections using a 5 μm ID capillary. The increase in sensitivity with small capillary diameters can be primarily attributed to a reduced mass flow rate of buffer and other background constituents into the electrospray source, which allows for greater sample ionization efficiency. A model that qualitatively accounts for the results is presented, but quantitative agreement is precluded due to difficulties in accounting for contributions due to a liquid sheath flow used with the electrospray source. The model accounts for the observation that the ESI/MS appears to function as a concentration‐sensitive detector under many conditions using large‐diameter capillaries. A transition occurs, however, to a regime where the ESI/MS functions as a mass flow‐sensitive detector for small‐diameter capillaries, where the ESI current is limited by the rate of delivery to the ESI source of charge carrying species in solution. These results suggest peptide and protein analysis at low attomole and subattomole levels should be obtainable with alternative types of mass spectrometers.
Using capillary electrophoresis in the optimization of a carboxypeptidase Y catalyzed transpeptidation reaction
Authors: Vinther Anders , Adelhorst Kim , Kirk Ole
Published: May 2005

A peptide amide, R‐Arg‐NH2, was produced by carboxypeptidase Y (CPDY)‐catalyzed transpeptidation of a peptide, R‐Ala‐OH in presence of a large excess of Arg‐NH2. Baseline separation of R‐Ala‐OH and R‐Arg‐NH2 was achieved by free solution capillary electrophoresis (CE) analysis. With CE the reactions could be closely followed with an analysis frequency of 3–6 h−1. Due to a low consumption of sample per CE analysis (1–5 nL introduced, 5–6 μL in the sample vial), the reactions were performed in 100 and 250 μL volumes. Consequently, the optimization experiments consumed limited amounts of enzyme and substrate only. At optimized experimental conditions approximately 90% conversion of the starting peptide, R‐Ala‐OH, to R‐Arg‐NH2 was achieved.
A model for the mobility of single‐stranded DNA in capillary gel electrophoresis
Authors: Luckey John A. , Smith Lloyd M.
Published: May 2005

The electrophoretic mobility of single‐stranded DNA in denaturing polyacrylamide gel‐filled capillaries is analyzed as a function of the applied electric field. The resultant mobility plots are complex functions of the fragment size and electric field. Traditionally, these plots are separated into three mobility regimes corresponding to Ogston sieving, reptation without stretching and reptation with stretching theories. However, none of these theories accurately models the variations in mobility that we observe with electric field strength. As a result, we propose a modification of the Ogston sieving theory which accounts for the stretching of migrating DNA molecules in the direction of the electric field. This theory assumes that the applied electric field in conjunction with the gel matrix distorts the DNA, altering the effective size of the migrating molecule. The stretched DNA offers a smaller cross‐section to the gel pore and thus sieves as though it were a smaller molecule. In this modified Ogston theory, the electrophoretic mobility depends only on the applied electric field, the size of the fragment, and constants which are independent of size and field strength. The modified Ogston equation accurately predicts the mobilities of DNA fragments in all three mobility regimes9 providing a single, simple model to account for all of the observed behavior.
Capillary zone electrophoresis of large DNA
Authors: Guszczynski Tadeusz , Pulyaeva Helena , Tietz Dietmar , Garner Mark M. , Chrambach Andreas
Published: May 2005

Capillary zone electrophoresis (CZE) of DNA 23.1 to 48.5 kb in length in polyacrylamide solutions of several concentrations provides evidence for polymer concentration and DNA length‐dependent stretching and orientation of these species and suggests an effective separation at a polymer concentration of about 0.6%. Applying a 0.1 % polyacrylamide concentration to the λ‐phage DNA ladder, at least 5 components are separated; separation improves with lowering of the field strength to 2 V/cm and, correspondingly, extended duration of CZE. Saccharomyces pombe chromosomal DNA separates into 3 major components on CZE at high field strength (270 V/cm) in 0.9% polyacrylamide solution, confirming a previous finding made on electrophoresis in a 1.1 mm ID tube at low field strength. However, the finding is limited to one source of the DNA plug, and the chromosomal identity of the components remains unknown. Methodological problems in the CZE of large DNA relate to the need for extended duration of pressure injection if absorbance detection is applied, the need to define the starting zone after extended pressure injection, the need to melt and digest agarose plugs prior to loading, and related needs for thermostating of the sample chamber and for software compatible with low voltage operation.
Capillary electrophoresis/mass spectrometry determination of inorganic ions using an ion spray‐sheath flow interface
Authors: Huggins Thomas G. , Henion Jack D.
Published: May 2005

The determination of inorganic cations and anions by capillary electrophoresis/mass spectrometry (CE/MS) is reported using an ion spray‐sheath flow interface coupling. A twelve‐component synthetic mixture of cations which included the positive ions of K, Ba, Ca, Mn, Cd, Co, Pb, Cr, Ni, Zn, Ag, and Cu was loaded into the capillary column at levels ranging from 30 to 300 pg, separated by CE, and detected by indirect UV and in the full‐scan (m/z 35–450) positive ion CE/MS mode using an aqueous buffer containing 30 mM creatinine and 8 mM α‐hydroxyisobutyric acid, pH 4.8. Creatinine forms adducts with the cations which are observed in the gas phase and requires rather high (120 electron volts) declustering energy to dissociate. This produces a reduction in charge state to form the free, singly charged, inorganic cations which are observed in the mass spectra. CE/MS analysis of an aqueous acidic extract of used aircraft engine oil revealed high levels of lead as well as lower levels of chromium and nickel. CE‐indirect UV analysis of a synthetic mixture containing 300 pg each of 11 inorganic ions, which included the anions of Br, Cl, NO2, NO3, S2O3, N3, SCN, SO4, SeO4, oxalate, and MoO4, is shown. The running buffer which affected this separation contained 5 mM ammonium dichromate, 10 mM ammonium acetate, and 20 mM diethylenetriamine at pH 9.3. Although indirect UV detection revealed good separation of these anions, CE/MS analysis of this mixture was complicated by interfering ion current signals from the cluster ions formed by the interaction between the additives and the analytes. Thus only three of these singly charged anions, e.g. Br, SCN, and HSeO4, could be satisfactorily detected in this mixture by CE/MS.
Acetylator phenotyping via analysis of four caffeine metabolites in human urine by micellar electrokinetic capillary chromatography with multiwavelength detection
Authors: Guo Ruichen , Thormann Wolfgang
Published: May 2005

The simultaneous analysis of 5‐acetylamino‐6‐formylamino‐3‐methyluracil (AFMU), 5‐acetylamino‐6‐amino‐3‐methyluracil (AAMU), 1‐methylxanthine (1X) and 1‐methyluric acid (1U), the four urinary metabolites of caffeine employed for acetylator phenotyping in man, by micellar electrokinetic capillary chromatography (MECC) is reported. Using on‐column fast scanning multiwavelength detection is shown to permit unambiguous identification of AAMU, AFMU, 1X and 1U in the three‐dimensional electropherograms obtained with both direct urine injection or application of extracted urine. The phenotype status of a number of healthy volunteers, some with known acetylator status, were determined using peak height ratios of AFMU/1X, (AFMU + AAMU)/1X and compared to those obtained by high performance liquid chromatography (HPLC) employing the AFMU/1X peak height ratio. Another set of MECC data was obtained after deliberate conversion of AFMU to AAMU, the results being expressed by AAMU/1X, AAMU/(1U + 1X) or AAMU/(AAMU + 1U + 1X) ratios. The data suggest that most reliable data are obtained when phenotyping is conducted under conditions of complete conversion of AFMU to AAMU. Phenotyping employing caffeine and analysis of its urinary metabolites by MECC is shown to be simple and, with automated instruments, potentially suitable for routine use and large‐scale population analysis. The agreement of MECC data obtained from three different instruments demonstrates the high level of maturity of this emerging technology.
On the use of dimensionless parameters in acid‐base theory. III. The molar buffer capacity of trivalent protolytes
Authors: Rilbe Harry
Published: May 2005

The general equation for the buffer capacity of trivalent weak acids is derived by way of the mean valence. It is valid for trivalent bases and ampholytes as well. Special interest is devoted to protolytes with symmetrically spaced pK values since both phosphoric acid and citric acid have this property. The symmetry implies a mathematical simplification which makes it possible to deduce the locations and magnitudes of the maxima and minima. Thus it is shown that the buffer capacity curve has three maxima and two minima for ΔpK values bigger than 1.29.
Quantitative analysis of gel electrophoretograms by image analysis and least squares modeling
Authors: Vohradský Jiří , Pánek Josef
Published: May 2005

A computer‐aided quantitative method for a complex analysis of gel electrophoretograms is presented. The analysis consists of several steps: (i) determination of the background image by methods of mathematical morphology and its subtraction from the gel image, (ii) selection of an appropriate part of the gel lane including curved lanes and lanes with a nonuniform width, (iii) computation of the lane densitogram by averaging several lane‐parallel scans, (iv) decomposition of the lane densitogram into component bands using a data selecting algorithm and Marquardt's minimizer. Several different functions for component bands are utilized. It is shown that the densitogram can be decomposed into component bands with reasonable accuracy only if an appropriate model function is chosen. The algorithms are tested on several different gel electrophoretograms which show typical features as a nonuniform background, curved lanes, an asymmetrical band shape and a superposition of small bands on the shoulders of big ones. It is shown that overlapped bands are best approximated by an asymmetrical Gaussian curve and an asymmetrical Gauss‐Cauchy function. Linear response to the serial dilution of the protein sample is tested.
Towards stoichiometric silver staining of proteins resolved in complex two‐dimensional electrophoresis gels: Real‐time analysis of pattern development
Authors: Rodriguez Lewis V. , Gersten Douglas M. , Ramagli Louis S. , Johnston Dennis A.
Published: May 2005

Toward the ultimate goal of deriving quantitative protein data from silver‐stained proteins resolved in complex two‐dimensional electrophoresis gels we have performed computerized real‐time analysis during pattern protein “silver stain” development. Three points emerged from this study: (i) Development time is a far more important variable than originally envisioned. (ii) Previous studies which, on the basis of a single development time, sought to relate silver stain slope and threshold properties to amino acid composition are non‐informative since it will be shown that slope values change markedly with development time, (iii) The combination of real‐time analysis and total protein load consideration allows, for the first time, protein “spots” to be grouped into categories, which in turn aid in quantification.
Characterization of proteins by capillary electrophoresis in fused‐silica columns: Review on serum protein analysis and application to immunoassays
Authors: Chen Fu‐Tai A. , Sternberg James C.
Published: May 2005

Protein mixtures can be characterized in terms of their separations by capillary electrophoresis (CE). The separation of proteins by CE is performed in untreated fused‐silica columns. Model proteins and complex protein mixtures with pI values ranging from 4.0 to 11.0 are separated in such columns in less than 10 min in the presence of phosphate buffer with a pH between 4.0 and 9.0. The application of CE separation procedures for routine analysis of protein in serum, urine, and cerebrospinal fluid in borate‐based buffer is also demonstrated. The detection of protein in CE is usually based on the intrinsic ultraviolet (UV) absorbance of the peptide bond at or near 200 nm, which provides a detection limit of about 10−5 M. The same protein separation procedures can also be applied to immunochemical reaction systems in which one component is labeled. Thus, an antigen analyte, or the antibody to the analyte, may be labeled with a fluor and detected by laser‐induced fluorescence (LIF). With a fluorescent‐labeled reactant, the use of LIF detection further extends the detection limit to 10−11 M. The CE separation technique for proteins provides a means to separate the bound and free species of the labeled antigen or antibody without the use of a solid support. The application of these separation techniques in conjunction with laser‐induced fluorescence detection to make possible the homogeneous immunochemical measurement of species at concentrations in the range of 10−9 to 10−10 M is shown.
Application of dynamic capillary isoelectric focusing to the analysis of human hemoglobin variants
Authors: Molteni Sarah , Frischknecht Hannes , Thormann Wolfgang
Published: May 2005

Capillary isoelectric focusing (CIEF) with electroosmotic zone displacement of normal and pathological hemoglobins (Hb) is reported. CIEF is performed in untreated, open‐tubular, fused‐silica capillaries of 75 μm internal diameter using methylcellulose for dynamic column conditioning. After direct injection of hemolysates mixed with carrier ampholytes, high resolution separation of Hb variants, including Hb A1c, A, F, D, S, E and A2, is obtained, this permitting unambiguous characterization of Hb patterns of normal adults, newborns, patients with diabetes, different hemoglobinopathies and thalassemia syndromes. Qualitatively, the CIEF data compare well with those obtained by gel isoelectric focusing and high‐performance liquid chromatography. CIEF is demonstrated to be a simple, rapid and fully instrumental approach to Hb analysis. Run times of less than 20 min make CIEF an attractive method for routine Hb investigations and screening programs.
Separation and sequencing of familiar and novel murine proteins using preparative two‐dimensional gel electrophoresis
Authors: Merrick B. Alex , Patterson Rachel M. , Witcher Lora L. , He Chaoying , Selkirk James K.
Published: May 2005

Strategies are needed for rapid protein isolation in order to identify disease‐related proteins and facilitate the design of oligonucleotides for further molecular inquiry. In our laboratory, C3H10T1/2 murine fibroblasts have been found to express a variety of proteins in various subcellular fractions which are relevant to experimental transformation and carcinogenesis. Preparative two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) procedures were developed to identify major cytoplasmic proteins by electroblotting and microseqencing. Isoelectric focusing tube gels were enlarged to 6 mm ID to accommodate larger protein loads at 0.5 to 2 mg protein. Separated proteins were electrotransferred from 6 mm thick slab gels onto 0.22 μ polyvinylidene difluoride membranes. Nearly 100 prominent blotted proteins were stained with Coomassie Brilliant Blue between pI 4.5–7.0 and 18–106 kDa and, of these, 27 prominent and well‐resolved proteins were selected for sequencing. Sequences of 14 to 24 amino acid residues in length were obtained from 11 proteins which were identified from computerized databases. Some of these identified proteins had structural or enzymatic functions while others had only recently been discovered, including a newly reported Hsp 70 class member and a novel calcium‐binding protein, reticulocalbin. The new heat shock protein has a molecular mass of 75 kDa and has been designated as Grp75, PBP74, CSA or p66mol‐1 in mice and humans with purported roles in transformation and antigen processing. Reticulocalbin is an endoplasmic reticular protein which contains six domains of the EF‐hand motif associated with high‐affinity calcium‐binding proteins. It may be involved in protein transport and luminal protein processing. In addition, sequences of 5 to 11 residues in length were also obtained from six other unidentified proteis. Thus, we have found that preparative 2‐D PAGE serves as a powerful one‐step purification method for protein isolation and characterization from an important in vitro murine model for the study of carcinogenesis.
Improved separation of diastereomeric derivatives of enantiomers by a physical network of linear polyvinylpyrrolidone applied as pseudophase in capillary zone electrophoresis
Authors: Schützner Wolfgang , Caponecchi Gabriele , Fanali Salvatore , Rizzi Andreas , Kenndler Ernst
Published: May 2005

Diastereomeric analytes were separated using capillary zone electrophoresis with polyvinylpyrrolidone as polymeric additive to the buffer solution. As test substances derivatives of D‐ and L‐ α‐amino and α‐hydroxy acids formed by reaction with (+)‐O,O′‐diacetyl‐ and (+)−O,O′‐dibenzoyl‐L‐tartaric anhydride, respectively, were used. The physical network formed by the linear polymer is supposed to act as a kind of pseudophase. It was found that the network affects the mobility of diastereomeric compounds to a different extent, enchancing the selectivity of the system. In nearly all cases of aromatic acids the diastereomer carrying the D‐amino acid was more strongly retained than the L‐isomer, as opposed to the situation with aliphatic acids.
About some aspects of the use of charged cyclodextrins for capillary electrophoresis enantioseparation
Authors: Chankvetadze Bezhan , Endresz Gabriele , Blaschke Gottfried
Published: May 2005

Free capillary zone electrophoresis with the negatively charged polyanion of the β‐cyclodextrin sulfobutyl ether (SBE‐β‐CD) as a chiral additive was used for the resolution of basic racemic drugs. High enantioselectivity was established for some racemic compounds using extremely low (micromolar) concentrations of the chiral additive. The dependencies of the migration times and the selectivity of the enantioseparation on the concentration of the chiral additive and the pH of the run buffer were studied. Examples of the chiral separation in counter‐current flows of discrete zones of the chiral selector and the racemic compound as well as separation of the neutral racemic compound thalidomide in a micellar electrokinetic chromatography‐like modus were demonstrated using SBE‐β‐CD.
Enantiomeric separation of salbutamol and related impurities using capillary electrophoresis
Authors: Rogan Manus M. , Altria Kevin D. , Goodall David M.
Published: May 2005

The enantiomeric resolution of salbutamol and its chiral and achiral related impurites is investigated using capillary electrophoresis. The effects of nine varieties of cyclodextrin, cyclodextrin concentration and organic modifier concentration were studied in an attempt to resolve all possible analytes in a complex mixture of salbutamol‐related solutes. Eleven components including three enantiomeric pairs were baseline resolved using 112 mM dimethyl‐β‐cyclodextrin at pH 2.5 in a citric acid/phosphate buffer. Both methanol and iso‐propyl alcohol at up to 20% were found to have a deleterious effect on the separation. Binding constants and mobility values for the free and complexed forms for each solute were determined. The results are interpreted by considering the physical properties of the molecules under the conditions employed and a rationale proposed for the underlying basis for chiral and achiral selectivity.
Quantitation in chiral capillary electrophoresis: Theoretical and practical considerations
Authors: D'Hulst Annick , Verbeke Norbert
Published: May 2005

Capillary electrophoresis (CE) represents a decisive step forward in stereoselective analysis. The present paper deals with the theoretical aspects of the quantitation of peak separation in chiral CE. Because peak shape is very different in CE with respect to high performance liquid chromatography (HPLC), the resolution factor Rs, commonly used to describe the extent of separation between enantiomers as well as unrelated compounds, is demonstrated to be of limited value for the assessment of chiral separations in CE. Instead, the conjunct use of a relative chiral separation factor (RCS) and the percent chiral separation (%CS) is advocated. An array of examples is given to illustrate this. The practical aspects of method development using maltodextrins – which have been proposed previously as a major innovation in chiral selectors applicable in CE – are documented with the stereoselective analysis of coumarinic anticoagulant drugs. The possibilities of quantitation using CE were explored under two extreme conditions. Using ibuprofen, it has been demonstrated that enantiomeric excess determinations are possible down to a 1% level of optical contamination and stereoselective determinations are still possible with a good precision near the detection limit, increasing sample load by very long injection times. The theoretical aspects of this possibility are addressed in the discussion.
Separation of α‐hydroxy acid enantiomers by high performance capillary electrophoresis using copper(II)‐L‐amino acid and copper(II)‐aspartame complexes as chiral selectors in the background electrolyte
Authors: Desiderio Claudia , Aturki Zeineb , Fanali Salvatore
Published: May 2005

Optical isomers of some α‐hydroxy acids, namely 2‐, 3‐phenyllactic acid, mandelic, p‐hydroxy‐, m‐hydroxy and 3,4‐di‐hydroxymandelic acid, were separated by means of capillary zone electrophoresis in free solution, using copper (II) complexes with L‐amino acid or aspartame ligands in the background electrolyte. The concentration and the pH dependence of the enantiomer separations have been studied in the cases of different chiral ligands and/or analytes. With the use of L‐proline as ligand only the optical isomers of 3‐phenyllactic acid were resolvable, whereas using L‐hydroxyproline the D and L forms of all compounds, except for 2‐phenyllactic acid, were separated. Better results were obtained with aspartame as chiral ligand.

Part of this work was presented at the 5th International Symposium on High Performance Capillary Electrophoresis, January 25–28, 1993, Orlando, FL, USA.
A polymerase chain reaction‐based method for the identification of DNA samples from common vertebrate species
Authors: Hershfield Bennett , Chader Gerald , Aguirre Gustavo
Published: May 2005

Polymerase chain reaction (PCR) amplification of vertebrate genomic DNAs using a (CAC)n primer was found to generate species‐specific patterns which are resolvable by agarose gel electrophoresis. Of the thirteen species tested (trout, frog, chicken, mouse, rat, cow, dog, African green monkey, chimpanzee, orangutan, gorilla, rhesus macaque, and human), all species showed discrete amplification products ranging in size from 1.0 to 3.2 kbp although trout and frogs had only weak (CAC)n amplification products. Most species had three to six bands except rodents, who had eight bands, and cows, who had a single band at 2.2 kbp. Importantly, within a single species, different unrelated individuals had highly similar amplification band patterns although the relative intensities of the bands varied. This held true even when different unrelated humans of different racial backgrounds were tested. We conclude that this technique is potentially useful for identifying which vertebrate species contributed DNA to a given biological sample (e.g., bloodstains, semen, hair) and that only a very small amount of sample is necessary for the analysis.
Detection of lectin activity on western blots using erythrocytes
Authors: Rao Ummiti J. S. Prasada , Ramasarma Pallavur R. , Rao Desiraju Rajagopal , Prasad Kanteti V. S.
Published: May 2005

A method is described for the direct detection of lectins, agglutinating erythrocytes, on nitrocellulose membranes after Western blotting, thus avoiding protein extraction from specific bands in the gel, followed by agglutination assays. The methodology essentially involves exposing the lectin band on a nitrocellulose strip to trypsinized rabbit erythrocytes (2%, in 0.15 M NaCl) for 30 min at 37°C and then carefully transferring the membrane to saline (4°C) for a few gentle washes and then fixing it in a solution (0.2% glutaraldehyde in 0.15 M NaCl) for 30 min. Later, the membrane is gently washed several times in 0.15 M NaCl containing 10 mM β‐alanine. The lectin band is visualized as a red agglutinated patch. The method is specific for lectins that can agglutinate red blood cells and virtually has no cross reactivity with the various nonlectin proteins tested. Binding of erythrocytes to the lectin band on the nitrocellulose strip can be prevented by specific competing sugars. The method can be applied to screen for the presence of lectins in natural materials and to monitor lectin fractions during purification.
Pulsed‐field gel electrophoresis for the separation of large protein molecules exemplified by the isoforms of apolipoprotein (a)
Authors: Csako Gyorgy , Nagy Balint , Costello Rene , Castelli Joann C. , Hruszkewycz Andrew M.
Published: May 2005

The performance of pulsed‐field gel electrophoresis (PFGE) was assessed for the separation of protein molecules. The allelic isoforms of apolipoprotein (a) (apo[a]) served as a model for this study because apo(a) is an unusually large protein, consisting of a variable number of repeating units. PFGE and, for comparison, conventional electrophoresis of human sera were carried out under reducing conditions in agarose gel. After blotting proteins onto nitrocellulose membrane, a combination of monospecific rabbit anti‐apo(a) antibody and alkaline phosphatase‐conjugated protein A detected apo(a) isoforms in all sera tested. The various apo(a) isoforms were effectively resolved within two repeating units (“kringles”) by both PFGE and conventional electrophoresis, but the type of agarose gel used greatly affected the speed of electrophoretic separation. In a series of 89 human sera, 59 double‐band and 30 single‐band patterns were seen using either electrophoretic system. However, one specimen produced double bands with PFGE, while only a single band could be detected by conventional electrophoresis, and with another specimen the opposite occurred. A total of 22 different apo(a) isoforms were identified among these patterns. It is concluded that the increasingly available PFGE technology is a practical alternative to conventional agarose electrophoresis for the separation of large protein molecules.
Capillary zone electrophoresis determination of phenylalanine in serum: A rapid, inexpensive and simple method for the diagnosis of phenylketonuria
Authors: Tagliaro Franco , Moretto Sara , Valentini Roberta , Gambaro Greta , Antonioli Claudio , Moffa Manuela , Tató Luciano
Published: May 2005

A simple, rapid, and quantitative capillary zone electrophoresis method for phenylalanine analysis in serum has been developed, with the aim of providing an analytical tool, as an alternative to liquid and gas chromatography, for the routine laboratory diagnosis of phenylketonuria. Electrophoresis was carried out in a 65 cm long, 50 μm wide bare silica capillary, using 0.025 M borate (adjusted to pH 10 with 1 M NaOH) at a potential of 20 kV, with in‐column UV detection at 214 nm. Under these conditions, the three aromatic amino acids (tryptophan, phenylalanine and tyrosine) migrated according to the pKs of the respective amine (and hydroxyl) groups. The efficiency of separation was about 150000 plates/column for phenylalanine. Diprophylline was adopted as internal standard. The injection of ethanoldeproteinized normal control serum gave rise to only a few major peaks not interfering with phenylalanine; phenylalanine in serum at normal concentrations appeared in a clean region of the electropherogram as a symmetrical peak with a migration time of about 11 min. The sensitivity was ≥ 3 μg/mL, with s/n ratio = 3. The linearity, in the range of 5–175 μg/mL, was described by the equation y 1.407–0.583 x, r2 = 0.9998. Accuracy and precision were satisfactory, with intra‐day and inter‐day coefficients of variation lower than 4% and 7%, respectively. The injection of sera from five phenylketonuria patients gave electropherograms clearly showing huge peaks of phenylalanine, thus allowing an easy laboratory diagnosis of phenylketonuria.

A preliminary version of this paper was presented at the 17th International Symposium on Column Liquid Chromatography, HPLC '93, Hamburg, May 9–14, 1993.
Data management in a two‐dimensional gel electrophoresis laboratory: “gelscript 1” software
Authors: Rovensky Pavol , Lefkovits Ivan
Published: May 2005

We have developed a software package “gelscript 1”, to perform data operations in a laboratory that runs and analyzes a large number of two‐dimensional (2‐D) gels. This system is managed automatically by the “4th Dimension” relational database on a Macintosh platform. A guiding principle in the development of “gelscript 1” was that any piece of information is entered into the system only once, and is retrieved, copied, inserted or checked whenever required. In addition to saving labor, this principle ensures consistency and integrity of the database. The system always “knows” how many gels were run from any one sample, how long the films were exposed or were supposed to be exposed. It keeps track of data throughout all operations, warns of improper sample assignments, and prints labels for films, samples, protocols, etc. The software can be adapted to other applications, in which many samples are processed and where cross‐referencing is needed.
Detection of thymopoietin‐responsive proteins in nude mouse spleen cells by two‐dimensional polyacrylamide gel electrophoresis and image processing
Authors: Toda Tosifusa , Ishijima Yasushi , Matsushita Hiroshi , Yoshida Mitsutaka , Kimura Narimichi
Published: May 2005

Lymphoid cells isolated from the spleen of BALB/c nu/nu nude mice were treated with synthetic human thymopoietin, and newly synthesized proteins were labeled by [35S]methionine incorporation. In the control experiment, the same lot of spleen cells were incubated in the labeling medium without the addition of thymopoietin. Urea/detergent‐soluble proteins were extracted from the cells after 3 h incubation to be separated by two‐dimensional polyacrylamide gel electrophoresis. Spots of [35S]methionine‐labeled proteins were visualized by autoradiography and analyzed by image processing. The computer‐aided spot matching screened out three major thymopoietin‐responsive proteins, TRP‐1, −2 and −3. [35S]Methionine incorporation into TRP‐3, of which the isoelectric point and molecular mass were approximately pI5 and 10 kDa, respectively, was decreased by the thymopoietin treatment. In contrast with the down regulation, TRP‐1, which was slightly higher in pI and slightly larger in molecular mass, and TRP‐2, which was slightly higher in pI and almost the same in molecular mass as TRP‐3, were evidently induced by the treatment. However, TRPs could not be assigned to Thy‐1 antigen on the difference in molecular mass. The specific induction by the thymopoietin treatment suggested that TRP‐1 and −2 might be novel proteins related to the intracellular signal transduction.
Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: A practical approach to muscle contractile and regulatory proteins
Authors: Sobieszek Apolinary
Published: May 2005

Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first “big slab” system utilizes 18 × 20 × 0.1 cm3 gels and a 10–18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from “bottom to top” and 20% glycerol is added to the 18% acrylamide solution. The second “minislab” system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386–396), with 8 × 10 × 0.05 cm3 gels and 5–15% or 9–18% acrylamide gradient ranges. They are cast from “top to bottom” in 28‐piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle‐type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described.
Pulsed field electrophoresis for the separation of protein‐sodium dodecyl sulfate‐complexes in polyacrylamide gels
Authors: Houri André , Starita‐Geribaldi Mireille
Published: May 2005

Polyacrylamide gel electrophoresis of proteins was studied using a pulsedcurrent mode. A new “local field” distribution was used to correct the gel patterns and optimize migration. A corrective field was applied at fixed 2 s intervals to a constant field, inducing a complex relaxation mechanism. Calculated variations in the local field directions decreased the electric strain on the gel during the run, with resultant optimum gel structure. The relaxation mechanism was found to enhance the absolute mobility of proteins with shorter running times compared to constant field gel electrophoresis (CFGE) and other pulsed field techniques. The enhancement of molecular mobility was explored by transverse pore gradient gel electrophoresis. Ferguson curves which exhibited a convex shape in CFGE were linearized by the new pulsed‐field method named pulsed oscillatory high‐performance electrophoresis (POPE).
Analysis of the HLA‐DR gene locus by temperature gradient gel electrophoresis and its application for the rapid selection of unrelated bone marrow donors
Authors: Uhrberg Markus , Hinney Anke , Enczmann Jürgen , Wernet Peter
Published: May 2005

The human leucocyte antigen (HLA) class II compatibility of bone marrow donor and recipient is an essential prerequisite for the prevention of severe graft versus host disease and therefore for the successful outcome of bone marrow transplantation. In this study an efficient protocol was developed for the rapid analysis of the polymorphic HLA‐DR gene locus, based on DNA‐polymerase chain reaction (PCR) amplification of the variable exon II of the HLA class II DR genes and subsequent temperature gradient gel electrophoresis (TGGE). Computer‐assisted melting map calculations were carried out to determine the melting behavior of the different HLA‐DR fragments. Despite the high variability of the DR alleles on the nucleotide sequence level the calculations revealed a common melting domain structure of the different HLA‐DR fragments, which was experimentally confirmed by perpendicular TGGE. On parallel TGGE, all samples were separated under the same electrophoretical conditions using a single PCR fragment without GC‐clamp. TGGE was applied for the analysis of the DR alleles of numerous bone marrow donor/recipient pairs and compared to the corresponding serological and DNA typing results. The TGGE patterns were found to be different for all samples with different HLA‐DR typing results. Identical homoduplex and heteroduplex patterns occurred only in the case of complete genotypic HLA‐DR identity as determined by direct sequencing of PCR products.
Detection of a single base mismatch in double‐stranded DNA by electrophoresis on uncrosslinked polyacrylamide gel
Authors: Pulyaeva Helena , Zakharov Sergey F. , Garner Mark M. , Chrambach Andreas
Published: May 2005

Uncrosslinked polyacrylamide forms gels in the concentration range of 15–40% acrylamide. Electrophoresis in these gels of a commercially available 350 bp heteroduplex DNA preparation separates it from the homoduplex DNA of the same size. The separation is qualitatively equivalent to that previously achieved in a commercial proprietary gel (“Mutation Detection Gel” of AT‐Biochem), or in an equivalent 14 %T, 0.15%C (N,N′‐methylenebisacrylamide) gel, but the mechanical stability of mutation detection electrophoresis (MDE) gels or 0.15 %C gels is better than that of uncrosslinked polyacrylamide gels. The separation in any of these three gel media can be carried out in short gel tubes within a few hours of electrophoresis time. In both uncrosslinked polyacrylamide and MDE gel media, the Ferguson plots [log(mobility) vs. gel concentration] and the plots of effective molecular radius vs. gel concentration (“T‐plots”) of both the heteroduplex and homoduplex DNA indicate an augmented size but similar flexibility upon passage through the gel than exhibited by the components of a DNA standard ladder. Homoduplex and heteroduplex DNA correspondingly exhibit a parallelism of their Ferguson curves in transverse MDE pore gradient gel electrophoresis, suggesting a surface net charge difference, possibly due to a conformational reorientation too subtle to be detected by a shift in the slope of the Ferguson plot, as has been observed once previously with a “kinked” DNA species. The gel fiber radius or length per unit volume of uncrosslinked polyacrylamide and MDE gels do not differ significantly within confidence limits, which are wide compared to conventionally crosslinked gels, presumably because of their greater swelling.
The use of a hollow fiber membrane module in sample conditioning prior to electrophoresis
Authors: Schwarz Thomas , Sindern Susanne , Bartholmes Peter , Kaufmann Michael
Published: May 2005

A new method to continuously change the buffer conditions in samples prior to electrophoresis by counter‐current dialysis has been developed using a hollow fiber membrane module (VariPerm M, bitop, Witten). It is demonstrated that the collected fractions of a column eluate after hydrophobic interaction chromatography cannot be analyzed by sodium dodecyl sulfate‐polyac‐rylamide gel electrophoresis (SDS‐PAGE) due to the high content of ammonium sulfate. In contrast, when the hollow fiber membrane module is used for on‐line desalting of the eluate during chromatography, it is possible to detect distinct bands on SDS‐gels even after sample concentration by evaporation. Moreover, adding polyethyleneglycol 20000 to the counter‐current dialysis buffer the hollow fiber membrane module is suitable for combined on‐line sample desalting and sample concentration of living organisms during free flow electrophoresis (FFE): a culture of Nitrosomonas eutropha which could not significantly be detected by FFE without conditioning was successfully analyzed when concentrated and desalted on‐line during sample application.