bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nonlinear “Ferguson curves” by two runs of the commercial automated HPGE‐1000 gel electrophoresis apparatus with intermittent scanning of fluorescence
Authors: Yarmola Elena , Chrambach Andreas 
Published: May 2005

AbstractThe exploitation of gel electrophoretic migration distance to gain information of molecular and gel fiber properties depends on the functions relating mobility with gel concentration. To the degree that these are nonlinear, the definition of those functions by past methods has been excessively laborious or, in application of gel concentration gradients, based on a number of assumptions. The recent commercial introduction of gel electrophoresis apparatus capable of intermittent scanning of the pattern promised to solve these problems. The present study shows that such apparatus allows for a precise definition of a nonlinear “Ferguson curve” (mobility vs. gel concentration) in two experiments, using different gel concentrations in the eight channels of the HPGE‐1000 apparatus and 5–29 scans in each during the course of an electrophoretic run. Simultaneously, these Ferguson curves are obtained for five components of a DNA ladder ranging in DNA length from 121 to 1857 bp.
Rapid detection of toxoplasmic nucleic acid by enzyme‐linked immunofiltration‐assay after membrane transfer
Authors: Aubert Dominique , Lepan Herve , Lemaire Pascale , Foudrinier Frederique , Marx‐Chemla Cathy , Bonhomme Annie , Pinon Jean‐Michel 
Published: May 2005

AbstractWe report the use of enzyme‐linked immunofiltration‐assay (ELIFA) for rapid detection of Toxoplasma gondii DNA after transfer to nitrocellulose or nylon membranes. Saturation, specific labeling with a nonradioactive oligonucleotide probe and washing steps are included in an active filtration process, which is controled by microcomputer regulating reagent flow. This method provides excellent specificity, reproducibility and repeatability. Recycling in a closed circuit or by reversing the direction of flow provides the advantage of reducing the required volume of costly reagents (e.g. probes). ELIFA does not require lengthy membrane manipulation, and hybridization as well as stringency and revelation steps can be automated. The procedure can be carried out in less than 2 h, which is a major time saving compared to conventional solid‐phase molecular hybridization techniques.
On‐line capillary electrophoresis‐electrospray ionization mass spectrometry of nucleotides
Authors: Zhao Zhongxi (Zack) , Wahl Jon H. , Udseth Harold R. , Hofstadler Steven A. , Fuciarelli Alfred F. , Smith Richard D. 
Published: May 2005

AbstractCapillary zone electrophoresis (CZE) and reversed anionic capillary isotachophoresis (CITP) conditions have been developed for the separation of mixtures comprised of monophosphate nucleosides, pyridine and flavin dinucleotides, and monophosphate dinucleosides. Results for the on‐line coupling of CZE and CITP with electrospray ionization mass spectrometry (MS) are presented. CITP‐tandem MS is utilized to provide both molecular weight and structural information of monophosphate dinucleosides. The fragmentation pattern of dinucleotides in the low collision energy range is described. The resulting mass spectra are readily interpreted in terms of dinucleotide structures. These results demonstrate the new capability for applications for the study of DNA and RNA.
Separation and characterization of Arabidopsis thaliana proteins by two‐dimensional gel electrophoresis
Authors: Kamo Masaharu , Kawakami Takao , Miyatake Norifumi , Tsugita Akira 
Published: May 2005

AbstractArabidopsis (Arabidopsis thaliana) proteins were isolated from five tissues (leaf, stem, root, seed and callus), and separated by two‐dimensional gel electrophoresis (2‐DE). 2‐DE was carried out by immobilized pH gradient (IPG) in the first dimension, and by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the second dimension. With the aid of comigrated five‐marker proteins, the patterns of 2‐DE gels for each tissue were graphically combined by a computer into a single synthetic image for the integrated Arabidopsis protein spots. The protein spot images, altogether 4763, were characterized by both molecular mass and isoelectric point. Partial amino(N)‐terminal sequences of 101 protein spots were analyzed by Edman degradation. Fifty seven proteins were partially sequenced and 46 proteins appeared to have blocked N‐termini. Deblocking by hydrazine vapor was carried out on 14 proteins and two of them were found to be pyroglutamyl‐blocked N‐termini. Forty seven new proteins were found by the present investigation.
Two‐dimensional electrophoretic mapping of hepatic and renal stress proteins
Authors: Witzmann Frank , Clack James , Fultz Carla , Jarnot Bruce 
Published: May 2005

AbstractCellular stress proteins and molecular chaperones are responsive to a variety of stressors and therefore comprise an ideal set of proteins with the potential to be used as biomarkers of chemical toxicity. We have investigated the expression of a group of well established heat shock and glucose‐regulated proteins in the rat liver and kidney using large‐scale two‐dimensional protein electrophoresis and computerized image analysis. Our goal was to determine the level of their expression in unstressed target tissues and map their coordinate positions on conventional format two‐dimensional electrophoresis (2‐DE) gels. All the proteins studied, except for Hsp25 (heat‐shock protein) whose expression fell below the level of analyzable detection, were constitutively expressed in liver and kidney. With the exception of Hsp70, all the stress proteins analyzed were constitutively more abundant in the liver than the kidney. Comparison of the sum total of all stress protein abundances revealed a nearly threefold higher level of expression in the liver than the kidney. Our results suggest that this group of proteins has significant responsibilities in normal, unstressed cells, due to their constitutive abundance. Correspondingly, the 2‐DE stress protein pattern established in this study may be very useful in toxicologic screening as well as describing a broad range of molecular effects of xenobiotic exposure.
Effect of pH and ionic strength of running buffer on peptide behavior in capillary electrophoresis: Theoretical calculation and experimental evaluation
Authors: Cifuentes Alejandro , Poppe Hans 
Published: May 2005

AbstractThe effect of pH and ionic strength of running buffer on peptide behavior in capillary electrophoresis (CE) is studied. A system for predictions of peptide migration in CE (SPPMCE) developed in our laboratory has been tested in a wide range of pH and buffer concentrations. The SPPMCE consists of a computer program for calculating peptide pKa values, an equation which relates peptide structures to their electrophoretic mobilities and a coupled computer program for the prediction of electropherograms. More than 25 different buffers have been employed, covering a pH range of 2–11 and a concentration range of 5–100 mM. Results from experiments are compared with the theoretical predictions. Good agreement is observed, which confirms the utility of the SPPMCE and allows fast and easy optimization of peptide separations in CE, with nothing more than the amino acid sequence of the linear peptide as the input.
Determination of caseinomacropeptide with capillary zone electrophoresis and its application to the detection and estimation of rennet whey solids in milk and buttermilk powder
Authors: Van Riel Jan , Olieman Cornelis 
Published: May 2005

AbstractThe presence of rennet whey solids in milk powder and buttermilk powder could be detected by analyzing the caseinomacropeptide (6.7 kDa) content by capillary zone electrophoresis. A hydrophilically coated capillary was used in combination with 6 M urea in a citrate buffer at low pH. Under these conditions, genetic A and B variants migrated as a single peak. This afforded a detection limit of 0.4% of rennet whey solids in combination with large volume injections and on‐column isotachophoretic concentration of the sample. The detector response (UV at 214 nm) was linear in the range of 1–20% rennet whey solids and the recovery of caseinomacropeptide was 98%. Pseudo‐casei‐nomacropeptide, lacking the N‐terminal Met‐residue, could be separated from caseinomacropeptide, thus preventing false positive results for some types of acid buttermilk powder.
Performance of amino‐silylated fused‐silica capillaries for the separation of enkephalin‐related peptides by capillary zone electrophoresis and micellar electrokinetic chromatography
Authors: Thorsteinsdóttir Margrét , Isaksson Roland , Westerlund Douglas 
Published: May 2005

AbstractEnkephalin‐related peptides were separated at low pH in a capillary with covalently bonded aminopropyl groups. The peptides are electrostatically repelled from the capillary surface and much higher efficiencies and faster separations were achieved compared to separations using uncoated capillaries. At low pH the amino groups are protonated, which results in reversed electroosmosis. The influence of voltage and ionic strength on the mobility and the separation efficiency was studied. The repeatability of migration times within one day was very good with relative standard deviations of 0.3–0.7%. Increasing the pH decreased the electroosmosis, eventually turning towards the cathode in the pH range 5–6; the separation performance, however, was lower at higher pH. Neutral and anionic micellar agents were added to the background electrolyte at different concentrations; the enkephalins had weak association with the neutral micellar agents but were distributed to the anionic taurodeoxycholic acid (TDC) micelles, giving rise to changes in separation selectivities. Very high efficiencies were obtained for peptides with a low distribution to the TDC micelles, while the efficiencies were impaired for those with a strong association with the micelles, which may indicate a slow mass transfer in the association process.
On the separation mechanism of capillary sodium dodecyl sulfate‐gel electrophoresis of proteins
Authors: Guttman AndráS 
Published: May 2005

AbstractPolyethylene oxide‐mediated capillary sodium dodecyl sulfate‐electrophoresis is a recently established, high‐resolution separation method for fast purity check and molecular mass assessment of protein molecules. The effects of the sieving polymer chain length and concentration on the separation mechanism of sodium dodecyl sulfate‐protein complexes were examined. The studies aimed to clarify whether the separation can be described by either the Ogston sieving theory, or the reptation or reptation‐with‐stretching theory. Polyethylene oxides with molecular masses of 100000, 300000 and 900000 Da were used as separation matrices at various concentrations ranging from 1–4%, 0.5–2% and 0.25–1%, respectively. The separation phenomena was examined using a standard protein test mixture containing six proteins in the molecular mass range of 14200–97400 Da. A possible separation mechanism of reptation with stretching is suggested, where separation performance was improved with increasing sieving polymer chain lengths and/or concentration.
Multiplex reverse transcription polymerase chain reaction combined with temperature gradient gel electrophoresis as a tool for the normalized quantitation of intrinsic factor mRNA
Authors: Sölch Jens‐Peter , Arnold Georg J. 
Published: May 2005

AbstractFor the quantitation of intrinsic factor (IF) mRNA, an assay based on competitive reverse transcription and subsequent polymerase chain reaction (RT‐PCR) combined with temperature gradient gel electrophoresis (TGGE) was established and validated with respect to precision and accuracy. IF‐specific mRNA segments (“targets”) were coamplified with known amounts of homologous “standard” RNA molecules, which differed from the targets by one base substitution. Following amplification, TGGE heteroduplex analysis proved to be a powerful method facilitating the efficient separation of these nearly identical target and standard DNA products. The measured absolute copy numbers of IF mRNA were put into relation to the constitutively expressed mRNA specific for glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), quantified simultaneously by competitive multiplex RT‐PCR. The resulting normalized IF mRNA expression rate in terms of n copies of IF mRNA/copy of GAPDH mRNA is independent of the mRNA heterogeneity and the abundance of specific transcripts within the RNA population of interest. Therefore, normalization relative to the housekeeping gene GAPDH provides a widely applicable value for comparative studies of gene expression on the level of mRNA. Here, a normalized IF mRNA expression rate of three copies per GAPDH mRNA molecule was measured in human stomach mucosaEssential parts of this work were presented at the international meeting of the Electrophoresis Forum '94, Technical University Munich, October 24–26, 1994.
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Basics of quantitative polymerase chain reaction: 2. Electrophoresis and quantitation of polymerase chain reaction products
Authors: Sundfors Carola , Collan Yrjö 
Published: May 2005

AbstractThe performance of agarose and polyacrylamide (PAGE) gels in quantitating polymerase chain reaction (PCR) amplified c‐erbB2 and p53 gene sequences (213 and 133 base pairs, respectively) was studied by applying image analysis on photographed ethidium bromide stained gels. The 5 mm thick agarose gels were more insensitive than the 1 mm thin PAGE gels and already started to show saturation effects within a narrow concentration range. The explanation is the greater dilution of band‐associated products within the volume of the gel and the inefficient penetration of exciting UV light through the gel. Such effects produced inconsistency and dramatic variations in the band ratio estimates. In the system used by us, agarose electrophoresis and also electrophoresis using 5 mm thick PAGE gels have only a limited value in quantitation of PCR products with the band density ratios, but both can be used well in qualitative work. The far thinner (1 mm) PAGE gels, which did not markedly absorb UV light, performed better in the light of band density ratio estimates. The linear or approximately linear range of concentrations was wider. The coefficient of variation of band ratio, when estimated from the same gels, after loading them with aliquots of identical DNA from several PCR amplified tubes, was in the range of 4%. The absolute values of band densities had a more remarkable variation (than 4%) in both agarose and PAGE gels. Our recommendation is that quantitation of PCR products be done with band density ratio estimates, and in thin PAGE gels.
Eliciting macroporosity in polyacrylamide and agarose gels with polyethylene glycol
Authors: Charlionet Roland , Levasseur Laurent , Malandain Jean‐Jacques 
Published: May 2005

AbstractPreparation of highly porous polyacrylamide has recently been described (Righetti et al., Electrophoresis, 1992, 13, 587–595). In this report we add new observations on the conditions of promoting macroporosity in polyacrylamide gels and extend the possibility of eliciting this phenomenon to agarose matrices by the combined use of polyethylene glycol and glycerol. The process of cluster formation in hot agarose solution was studied and gel structures were examined by scanning electron microscopy. A definition of macroporosity in gel, related to controlled microsyneresis during gelation, is tentatively proposed. The unexpected influence of acrylamide and agarose concentrations upon the size of macroporous structures in the corresponding gels is revealed.
Application of gels of 0.5 mm thickness to electrophoresis in the automated HPGE‐1000 apparatus: Improved resolution
Authors: Chang Huan‐Tsung , Chrambach Andreas 
Published: May 2005

AbstractGel electrophoresis in commercial automated apparatus (HPGE‐1000, LabIntelligence, Menlo Park, CA) is conventially conducted in gels of 3 mm thickness at about 15 V/cm. Since the intermittent scanning of the gel allows one to measure band width as a function of migration time, resolution may be evaluated quantitatively. Comparing the value of resolution between two proteins in electrophoresis on agarose gels of 0.5 and 3.0 mm thickness and at various field strengths, it was found that within the given Joule heat dissipation capacity of the apparatus, resolution between the proteins is improved when the gel thickness is reduced from 3.0 to 0.5 mm, which allows for an increase in field strength from 15 V/cm, conventional for that apparatus, to 45 V/cm.
The influence of buffer composition on separation efficiency and resolution in capillary electrophoresis of 8‐aminonaphthalene‐1,3,6‐trisulfonic acid labeled monosaccharides and complex carbohydrates
Authors: Klockow Antje , Amadò Renato , Widmer H. Michael , Paulus Aran 
Published: May 2005

AbstractThe effect of buffer conditions — varying in salt type, pH, and concentration – on the separation of 8‐aminonaphthalene‐1,3,6‐trisulfonic acid (ANTS)‐labeled monosaccharides and complex‐type carbohydrates was investigated. Different buffer systems for high and low electroosmotic flow conditions were chosen: a phosphate and a citriate background electrolyte, each at pH 2.5, a phosphate buffer, pH 9.0, and a borate buffer at pH 9.5. All buffer systems displayed differences in resolution and selectivity. Phosphate and borate buffer demonstrated the greatest selectivity changes for ANTS‐labeled carbohydrates. While separation in the phosphate system relies mainly on differences in the charge‐to‐mass‐ratio, additional selectivity can be achieved with borate complexation of glycoconjugates. The use of borate buffers improved monosaccharide separations whereas complex carbohydrates showed a loss in resolution. The citrate background electrolyte at low pH caused no significant changes in the separation performance. The pH 9.0 phosphate buffer showed a reversed migration order of the ANTS conjugates with a decreased resolution, compared to the pH 2.5 phosphate buffer, due to the strong electroosmotic flow generated under high pH conditions. An ovalbumin‐derived oligosaccharide library demonstrates the significance of buffer selectivity for complex carbohydrate separations. The separation in the acidic phosphate and the alkaline borate buffer generates a different pattern and only the combination of both buffer systems allows an appropriate assessment of sample complexity.
Biotinylated hyaluronan: A versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure
Authors: Melrose James , Numata Yukiko , Ghosh Peter 
Published: May 2005

AbstractHyaluronan influences cellular proliferation and migration in developing, regenerating and remodelling tissues and in tissues undergoing malignant tumour‐cell invasion. The widespread occurrence of hyaluronan‐binding proteins indicates that the recognition of hyaluronan is important to tissue organisation and the control of cellular behaviour. A number of extracellular matrix and cellular proteins, which have been termed the hyaladherins, have specific affinities for hyaluronan. These include cartilage link‐protein, hyaluronectin, neurocan, versican and aggrecan, which all bind to HA within the extracellular matrix. Cellular receptors for hyaluronan such as CD44 and RHAMM (receptor for hyaluronate‐mediated motility) have also been identified. In the present study biotinylated hyaluronan (bHA) was prepared by reacting adipic dihydrazide with a 170 kDa hyaluronan sample using the bifunctional reagent 1‐ethyl‐3‐[3‐(dimethylamino) propyl] carbodiimide. The resultant free amine moeity of the hydrazido‐hyaluronan was then reacted with biotin succinimidyl ester (sulfo‐NHS‐biotin) to prepare the bHA. After 4–20% gradient sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and electroblotting to nitrocellulose membranes, bHA and avidin alkaline phosphatase conjugate could be used in conjunction with nitroblue tetrazolium/5‐bromo‐4‐chloro‐3‐indolyl phosphate substrates to specifically visualise with high sensitivity (≥2 ng), bovine nasal cartilage link‐protein, aggrecan hyaluronan binding region, and human fibroblast hyaluronan receptors such as CD‐44. Conventional Western blotting using specific monoclonal antibodies to these proteins was also used to confirm the identities of these proteins.
Pulsed electrochemical detection of nonchromophoric compounds following capillary electrophoresis
Authors: Lacourse William R. , Owens George S. 
Published: May 2005

AbstractPulsed electrochemical detection (PED) following capillary electrophoresis (CE) has been applied to the direct detection of polar aliphatic compounds. These compounds typically have poor optical detection properties and are considered to be electroinactive under constant applied potentials. The voltammetric responses at microelectrodes of model compounds were studied under various buffers and pH conditions. The detection of unsubstituted carbohydrates requires highly alkaline conditions, whereas amine‐containing compounds (e.g., glycopeptides, peptides, and amino acids) and thiocompounds can best be detected at mildly alkaline (i.e., pH 9.0) and mildly acidic (i.e., pH 5.5) conditions, respectively. In a comparative study of pulsed amperometric detection (PAD) and integrated pulsed amperometric detection (IPAD), IPAD is determined to be better suited to manage the large oxide‐formation backgrounds, which accompany oxide‐catalyzed detections of amine‐ and sulfur‐ containing compounds. IPAD results in more stable baselines, eliminates oxide‐induced artifacts, and often yields lower limits of detection. Mass detection limits under optimal conditions are typically 10 fmole or less. This paper delineates PED waveform parameters for the optimal detection of underivatized nonchromophoric compounds of biological significance in various operating buffers and electrolytes.
Capillary electrophoretic analysis of cyclodextrins and determination of formation constants for inclusion complexes
Authors: Lee Ying‐Haur , Lin Tsung‐I. 
Published: May 2005

AbstractCapillary zone electrophoresis (CZE) methods with indirect absorbance detection for analyzing mixtures of α‐, β‐, and γ‐cyclodextrins (CDs) and their derivatives have been developed. Benzylamine, salicylic, sorbic, or 1‐naphthylacetic acid (NAA) was utilized as background electrolyte (BGE) and absorbance provider. Separation of α‐, β‐, and γ‐CD could be achieved in less than 18 min when the CZE was run in 2 mM NAA or 5 mM sorbate solution (pH 12.2) and detected by indirect absorbance at 222 or 254 nm, respectively. Mixtures of α‐ and β‐CDs, and dimethyl‐ and trimethyl‐derivatives of β‐CD could also be analyzed by CZE, using 50 mM salicylic acid or benzylamine solution (pH 6.0) as BGE with indirect absorbance detection at 230 and 210 nm, respectively. CZE methods for determining the inclusion complex formation constants of various CDs for salicylic acid or benzylamine with either direct or indirect absorbance detection have also been developed. The formation constants of salicylate are in the range from ca. 8 ± 0.3 mole−1 for the complex with α‐CD to ca. 99 ± 2 molarity−1 for the complex with methyl‐β‐CD. The detection limits (determined at a signal‐to‐noise ratio of 3) for the NAA and the salicylate system are ca. 0.1 mM and 1 mM, respectively.