bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitation in chiral capillary electrophoresis: Theoretical and practical considerations
Authors: D'Hulst Annick , Verbeke Norbert 
Published: May 2005

AbstractCapillary 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

AbstractOptical 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

AbstractPolymerase 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

AbstractA 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

AbstractThe 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

AbstractA 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

AbstractWe 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

AbstractLymphoid 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

AbstractTwo 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

AbstractPolyacrylamide 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

AbstractThe 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

AbstractUncrosslinked 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

AbstractA 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.
Micellar electrokinetic capillary chromatography analysis of water‐soluble vitamins and multi‐vitamin integrators
Authors: Dinelli Giovanni , Bonetti Alessandra 
Published: May 2005

AbstractMicellar electrokinetic capillary chromatography (MECC) was applied to the determination of water‐soluble vitamins (WSV) in pharmaceutical preparations. The analytical procedure employed in the WSV separation by MECC showed a satisfactory average column efficiency (195000 N) and resolution (more than 100 theoretical peaks separable). The mean reproducibility in the retention time of consecutive and day‐to‐day runs was 1.1% and 5.1%, respectively. Analyses were performed after extraction of active ingredients by solid phase extraction. Recoveries (92–103%) and relative variation coefficients (less than 5%) confirmed the potential of MECC in the field of pharmaceutical analysis.
Isolation of human serum transferrin by free‐fluid recycling electrophoresis in simple buffers
Authors: Caslavska Jitka , Thormann Wolfgang 
Published: May 2005

AbstractThe isolation of human serum transferrin (Tf) using recycling isotachophoresis (RITP) and recycling isoelectric focusing (RIEF) with simple buffers is described. Serum fractionation, the first step in the protocol for Tf purification, is shown to be easily performed either by RITP of filtered serum using low molecular mass spacers or by RIEF of dialyzed serum employing a binary mixture of a well‐defined buffer pair covering the pH range between 5.2 and 6.2, called RIEF‐OptiFocus. For polishing, Tf‐containing fractions are reprocessed by RITP or RIEF‐OptiFocus. Other RIEF approaches based on the use of single amino acids at high concentration and ternary amino acid mixtures are shown to constitute less effective methods for Tf isolation. With a four‐step protocol, comprising in turn RITP, RIEF‐OptiFocus, ultrafiltration and again RITP, “single‐band” purity (as assessed by two‐dimensional gel electrophoresis) is obtained. Omission of the first step (RITP) and direct processing of dialyzed serum by RIEF‐OptiFocus, ultrafiltration and RITP, is shown to provide remarkable results as well.
Analysis of the inter‐alpha‐trypsin inhibitor polymorphism in West Africa: Description of a new allele, ITI*7
Authors: Caeiro José Luis B. , Liste Isabel , Vogt Ulrike , Ribeiro Joaquim C. Teixeira 
Published: May 2005

AbstractInter‐α‐trypsin inhibitor (ITI) phenotypes were classified in the West African population of Cabo Verde by polyacrylamide gel isoelectric focusing, followed by immunofixation and silver staining. Gene frequencìes of the alleles ITI*1, ITI*2, ITI*3 and ITI*4 were calculated to be 0.532, 0.153, 0.307 and 0.002, respectively. A new rare allele, ITI*7, was found, providing evidence for further genetic variability of the ITI protein. The ITI*7 allele frequency has been determined to 0.006. The assumption that allele ITI*3 may be used to characterize populations of African origin is supported by our data.
Mixed micellar electrokinetic chromatography of corticosteroids
Authors: Bumgarner Jefferson G. , Khaledi Morteza G. 
Published: May 2005

AbstractMixed micellar electrokinetic chromatography (MMEKC) using mixtures of bile salt surfactants and/or sodium dodecyl sulfate (SDS) was employed to separate a group of corticosteroids. Resolution in electrokinetic chromatography (EKC) can be greatly enhanced by the use of mixed micellar systems due to the fact that the composition of the mixed micelles has a great influence on retention, selectivity and the size of the elution window. By combining surfactants with different structural properties, solute‐micelle interactions were manipulated in order to elicit specific separations. Various combinations of bile salts and/or SDS at different mole fractions as well as total micelle concentrations were used in order to enhance the resolution of corticosteroid separations. Large changes in retention and selectivity were observed that often resulted in frequent variations in elution order. In addition, the composition of mixed micellar systems had a great influence on the elution window in EKC, as measured by the ratio of tmc/ teo. Addition of SDS to the mixtures of bile salt micelles resulted in significant extension of the elution window and subsequently improvement in resolution. A separation of seventeen corticosteroids was achieved. Finally, MMEKC was applied in order to separate the steroidal components of a mixture of three anti‐inflammatory creams.