bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Capillary 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

Arabidopsis (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

Cellular 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

The 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

The 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

Enkephalin‐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

Polyethylene 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

For 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 mucosa

Essential 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

The 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

Preparation 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

Gel 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

The 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

Hyaluronan 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

Pulsed 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

Capillary 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.
The investigation of glycosaminoglycan mimotope structure using capillary electrophoresis and other complementary electrophoretic techniques
Authors: Carney Stephen L. , Caterson Bruce , Penticost Helen R.
Published: May 2005

We have used various electrophoretic techniques to analyse glycosaminoglycan structure. Capillary electrophoresis has been particularly useful in the determination of the sulphation of glycosaminoglycans (GAG) and the sulphation of partly desulphated glycosaminoglycans produced by methanolysis. This, in conjuction with preparative electrophoresis and enzyme linked immunosorbent assay (ELISA) has permitted us to ascertain the length of the oligosaccharide required for binding and sulphate ester groups required for optimal binding and those essential for antibody binding. From these preliminary studies, we have demonstrated that the minimum length of oligosaccharide required for binding was about 12–14 monosaccharides in length. It appears likely that 6 sulphation is required for strong binding but 4 sulphation is not involved in mimotope binding. We propose on the basis of this evidence that the mimotope does not contain 4‐sulphate residues but 3‐4 6‐sulphate ester groups are essential for binding.
Purity of glycosaminoglycan‐related compounds using capillary electrophoresis
Authors: Malsch Reinhard , Harenberg Job
Published: May 2005

High performance capillary electrophoresis was used to determine impurities in glycosaminoglycans. The counterion of glycosaminoglycans was analyzed with indirect UV‐detection using a 40 mM 4‐aminopyridine buffer. Calcium, lithium, potassium and sodium could be resolved. A linear correlation between the area under the curve and the concentration of sodium (r2 = 0.98) and calcium (r2 = 0.99) was found. Using enzymatic depolymerization, chondroitin sulfates were cleaved to disaccharides. The resulting disaccharides, with the structure 4‐deoxy‐α‐L‐threo‐hex‐4‐enopyranosyl uronic acid (ΔUA) 2 × (1 → 3)‐D‐GalNY6X (X = H, sulfate and Y = acetyl, sulfate) for dermatan sulfate, were detected selectively at 230 nm using capillary electrophoresis. Dermatan sulfate disaccharides were analyzed using a 50 cm long fused silica capillary (75 μm ID). The buffer used was 10 mM sodium tetraborate and 50 mM SDS, pH 8.8. The detection was at 230 nm. Using the main peak ΔUA (1 → 3)‐D‐GalNAc4S as standard, between 1 and 80% dermatan sulfate in heparin preparations were analyzed. The disaccharide showed a linear correlation of the peak area versus the concentration with a correlation coefficient r2 = 0.98. The methods are useful in characterizing the identity and concentration of the counterion of glycosaminoglycans after chondroitinase degradation.
Electrophoretic separation of biopolymers in a matrix of polyacrylamide covalently linked to agarose
Authors: Chiari Marcella , Campoleoni Andrea , Conti Paola , Felli Chiara , Patrosso Maria C. , Brogren Carl H.
Published: May 2005

A new type of agarose polyacrylamide mixed‐bed gel, obtained by simultaneous gelation of a novel type of allyl‐activated agarose and its copolymerization with acrylamide, has pore sizes intermediate between those of polyacrylamide and agarose. The process used to activate the agarose chains enables the substitution to be controlled. As indicated by nuclear magnetic resonance (NMR), only one allyl group was inserted per agarose basic unit. Several formulations of mixed‐bed gels, containing different percentages of acrylamide, were compared with conventional polyacrylamide or agarose gels. Resolution, migration distance and band sharpness of different molecular mass fragments were evaluated, with two types of gel run side‐by‐side in a vertical or horizontal system. The faster electrophoretic mobility of DNA in dilute mix‐bed gels and the improved separation of the component of high molecular mass (1 to 6 kbp) of the 1 kbp ladder indicate that these matrices have larger porosity than any dilute polyacrylamide formulations. Sodium dodecyl sulfate (SDS)‐protein complexes migrate in the mixed gels faster than in polyacrylamide gels of the same %T.
Interaction of lectins with their ligand carbohydrate of α‐fetoprotein: Analysis by mixed‐lectin affinity electrophoresis
Authors: Taketa Kazuhisa , Liu Miao , Taga Hiroko
Published: May 2005

Serum or ascites α‐fetoprotein (AFP) from patients with hepatocellular carcinoma and from a cord blood were analyzed by affinity electrophoresis with two lectins mixed in agarose gel in a combination of concanavalin A (Con A) and Lens culinaris agglutinin A (LCA‐A) or of erythroagglutinating phytohemagglutinin (E‐PHA) and Allomyrina dichotoma lectin (allo A). Con A‐ and LCA‐A‐reactive AFP‐C2‐L3 was not further retarded by mixing with either of the other lectin. It showed a mobility identical with that of AFP‐C2 or AFP‐L3. E‐PHA‐ and allo A‐reactive AFP‐P4‐A3 showed similar results. It migrated with intermediate mobilities of AFP‐P4 and AFP‐A3 depending on the concentrations of the two lectins mixed in the gel. the results indicate that the two mixed lectins compete with each other for the topologically different lectin‐binding sites on the oligosaccharide of AFP molecule.
Identification of glycosylated forms of wheat storage proteins using two‐dimensional electrophoresis and blotting
Authors: Laurière Michel , Bouchez Isabelle , Doyen Christine , Eynard Lucia
Published: May 2005

Two‐dimensional electrophoresis with acid‐polyacrylamide gel electrophoresis (PAGE), followed by sodium dodecyl sulfate (SDS)‐PAGE and SDS‐PAGE of unreduced polypeptides followed by SDS‐PAGE under reducing conditions, were used to separate and identify the different subgroups of gliadins and glutenins and to distinguish between covalent and noncovalent polymers of glutenins. Gels were blotted under semidry conditions according to Laurière (Anal. Biochem. 1993, 212, 206–211) to allow large polymers of glutenins to be transferred efficiently. Glycosylated polypeptides were detected on blots using either the method of Haselbeck and Hösel (Glycoconjugate J. 1990, 7, 63–74), or using anti‐(xylose‐containing N‐glycan) antibodies (Laurière et al., Plant Physiol 1989, 90, 1182–1188). High and low molecular weight glutenin subunits were shown to aggregate through both disulfide bridges and noncovalent protein‐to‐protein interactions. Aggregated γ‐gliadins were also demonstrated. Glycans were detected on both gliadin and gluteinin polypeptides. Covalently aggregated low molecular weight glutenins were shown to contain N‐glycans with xylose, which demonstrated their sorting in the Golgi apparatus.
Soldium dodecyl sulphate‐polyacrylamide gel electrophoresis of human parotid salivary proteins: Comparison of dansylation, Coomassie Blue R‐250 and silver detection methods
Authors: Beeley Josie A. , Newman Frances , Wilson Paul H. R. , Shimmin Ian C.
Published: May 2005

Detection of human parotid salivary proteins by dansylation and UV‐transillumination after sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) has been compared with Coomassie Blue R‐250 and silver staining procedures. Dansylation gives superior results in terms of both resolution and sensitivity, especially with basic proline‐rich proteins (PRPs).
Electrophoretic fingerprinting and multiparameter analysis of cells and particles
Authors: Budde Axel , Knippel Eberhard , Grümmer Gerald , Treichler Jens , Brockmann Hannes , Donath Edwin , Bäumler Hans
Published: May 2005

A recently developed electrophoretic instrumentation based on a real‐time image processing system has been applied to electrophoretic fingerprinting and multiparameter analysis of cells and other particles. The comparison between theoretical and experimental electrophoretic fingerprints, completed by the analysis of differences between measured fingerprints, offers a new methodology for better understanding and controlling of the processes at solid/liquid interfaces. Moreover, the multi‐parameter analysis including electrophoretic mobility, size, density and shape of cells can complete the diagnosis and prognosis of diseases.
Two‐dimensional electrophoretic patterns of acute‐phase human serum proteins in the course of bacterial and viral diseases
Authors: Bini Luca , Magi Barbara , Marzocchi Barbara , Cellesi Carla , Berti Brunilde , Raggiaschi Roberto , Rossolini Aldo , Pallini Vitaliano
Published: May 2005

Acute‐phase serum proteins were analyzed by two‐dimensional electrophoresis with isoelectric focusing in 3‐10 immobilized pH gradients. Most spots were identified by reference to the plasma map in the SWISS‐2DPAGE database. Serum amyloid A protein spots were identified by immunoblotting with specific antiserum and by matching determined with predicted values of electrophoretic parameters. Changes in the concentrations of α1‐antitrypsin, leucinerich glycoprotein, haptoglobin, serum retinol‐binding protein and transthyretin were quantitated by densitometry of silver‐stained gels. Electrophoretic patterns from 18 patients with bacterial diseases and 16 patients with viral diseases were compared. The incidence of serum amyloid A protein spots was 18/18 in bacterial diseases and 6/16 in viral diseases. As for the other reactants studied, variations were simultaneous in bacterial disease and tended to be staggered in viral diseases.
Electrophoretic analysis of biliary proteins: Application of high resolution two‐dimensional polyacrylamide gel electrphoresis with immobilized pH gradient in the first dimension
Authors: He Chang , Jüngst Dieter
Published: May 2005

Native bile samples, dialyzed against double distilled water, fractionated by Sepharose 2B column chromatography, concentrated by ultrafiltration and lyophilized, were solubilized in lysis buffer and studied by two‐dimensional electrophoresis (2‐DE) with immobilized pH gradients in the first dimension. We counted 70, and 7 spots in 2‐DE patterns of bile from patients with cholesterol gallstones and pigment gallstones, respectively.
Marker pattern instabilities as a major cause of reproducibility problems in two‐dimensional DNA fingerprinting
Authors: Hampe Jochen , Marczinek Karola , Preuss André , Nürnberg Peter
Published: May 2005

Two‐dimensional (2‐D) DNA fingerprinting is a promising technique for multilocus analysis of eukaryotic genomes. It has been successfully applied to the detection of DNA variation in tumors, to linkage analyses and to genomic comparisons of inbred mouse strains. However, there are still problems with inter‐gel comparisons of 2‐D DNA typing patterns as documented by the intergel reproducibility rates reported in the literature, which range from 84 to 98%. The basis for standardization in almost all of these studies has been a set of lambda fragments (digested separately with the restriction enzymes HaeIII, RsaI, Bg/I) that produces a spot pattern scattered across the gel. These spots are used as markers for gel comparisons. Since we noticed considerable variations in the marker spot paterns, we evaluated the properties of the lambda marker using both computer simulation and an empirical analysis of forty independent consecutive gels from our laboratory. We explain the instabilities of the spot pattern on the basis of the melting properties of the individual lambda fragments. A subset of spots is presented that has been stable in all our experiments. Only this set of spots should be used for gel standardization purposes until a new, completely reproducible marker becomes available. Finally, suggestions for an improved marker system are made.
Molecular analysis of Salmonella enterica serotype Dublin: Building bridges between population genetic and molecular epidemiological studies
Authors: Platt David J. , Browning Lynda M. , Candlish Denise
Published: May 2005

Population genetic studies of Salmonella enterica serotype Dublin using multilocus enzyme electrophoresis have recognised two dominant clones termed Du1 and Du3. The characterisation of plasmids in Dublin suggests greater strain diversity. The application of restriction enzyme fragmentation pattern (REFP) analysis of genomic DNA using Sau3A and HincII together with plasmid subtractive analysis can resolve anomalies in earlier comparisons. Twenty‐six isolates were selected for inclusion in the study. All had been previously characterised with respect to their plasmids, and were isolated from the USA, Canada and five European countries. On the basis of plasmid profiles, 17 were predicted to correspond with Du1 and Du3. Sau3A digestion generated two distinct REFPs (A and B) of < 70% similarity, which corresponded with Du1 and Du3. After the contribution of plasmid‐derived bands was subtracted, two variants of A (A1 and A2) and four of B (B1, B2, B3 and B4) were recognised. Seventeen were concordant with predictions from population genetic studies. Nine that could not be predicted on the basis of atypical plasmid profiles all showed REFP A1 (Du1) and were consistent with the incursion of additional plasmids or plasmid cointegration. REFPs from HincII digests generally corroborated Sau3A data but showed greater overall similarity between the strains and more influence from plasmid DNA

Presented at the British Electrophoresis Society Meeting, Maidstone Kent, UK, July 13–14, 1995.

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Improved conditions for the analysis of large variable number of tandemly repeated (VNTR) unit polymorphisms
Authors: Vandenbroeck Koen , Consiglio Antonella , Martino Gianvito , Grimaldi Luigi M. E.
Published: May 2005

Large variable number of tandemly repeated (VNTR) unit polymorphisms were identified by amplification of genomic DNA with appropriate primers, followed by agarose gel electrophoresis and ethidium bromide staining. However, when genomic DNA was extracted using paramagnetic beads, we found that the efficiency of polymerase chain reaction amplification of VNTR's was affected by the purity of the solid phase‐eluted DNA. Here we report that VNTR's located in the human interleukin‐1 receptor antagonist and interleukin‐4 genes are clearly and reliably amplified only when more stringent conditions are used in order to obtain purer DNA eluates from DNA‐bead complexes. In addition, polyacrylamide gel electrophoresis followed by silver staining is superior to ethidium bromide staining of agarose gels in visualizing the different allelic bands of heterozygous carriers.
From image processing to classification: IV. Classification of electrophoretic patterns by neural networks and statistical methods enable quality assessment of wheat varieties for breadmaking
Authors: Jensen Kristen , Kesmir Can , Søndergaard Ib
Published: May 2005

The end‐use quality of products made from doughs consisting of wheat flour and water is often dependent upon the storage (gluten) proteins of the grain endosperm. Today the electrophoretic patterns of the high molecular weight (HMW) glutenin subunits are used for quality selections in wheat breeding programs in several countries. In this study, we used two multivariate techniques to classify digitized patterns from isoelectric focusing of gliadins and glutenins: a two‐layered neural network architecture consisting of a self‐organizing feature map and a feed‐forward classifier [1], and discriminant analysis [2, 3]. Three groups of seven wheat varieties (Triticum aestivum L.), associated with poor, medium or good properties in relation to bread‐making quality, were used. The best classification results were otained by the neural network model, based on data from the gliadin fraction: it was possible to classify varieties associated with poor or good quality, with recognition rates of 70 and 69%, respectively. The statistical method was better suited to solve the classification problem when the data was based on the glutenin fraction: if a specific variety was already known to be non‐poor, this method enabled us to classify the medium‐ and good‐quality classes with recognition rates of 90 and 88%, respectively. The results obtained were confirmed by correlation coefficients.
Quantitation of the water channel protein aquaporin (CHIP28) from red blood cell membranes by densitometry of silver stained polyacrylamide gels
Authors: Benga Gheorghe , Banner Malcolm , Wrigglesworth John M.
Published: May 2005

A protein determination procedure which involves the densitometry of silver stained polyacrylamide gels is described. It involves calibration with bovine serum albumin and molecular weight markers on the same gel with the protein to be quantitated. The procedure is simple, rapid, reproducible and accurate and is more sensitive than other procedures for protein determination. The procedure is particularly useful in quantitating proteins purified in small amounts since the determination can be performed on the same gel used to check the purification. It avoids interference by detergents and other substances usually present in solutions of purified proteins. The procedure has been applied to the quantitation of a recently identified protein, aquaporin (CHIP28), assumed to be a major water channel in the red blood cell membrane. A quantitative analysis of a purified fraction of this protein shows that the 28 kDa component represents approximately two thirds of the protein content of the sample, with the remainder comprising a glycosylated, high molecular mass component. The procedure may be useful for quantitating proteins revealed on silver stained gels and could be included as a standard part of any protocol for protein purification.
The role of pH and membrane porosity in preparative electrophoresis
Authors: Corthals Garry L. , Margolis Joel , Williams Keith L. , Gooley Andrew A.
Published: May 2005

The Gradiflow is a preparative electrophoresis apparatus, allowing fractionation based on a combination of size and charge of proteins in their native (unreduced) form. The prepative fractionation of two proteins of similar size and isoelectric point is demonstrated using the Gradiflow. A separation membrance of appropriate pore size was chosen and then fractionation was “fine tuned” by selecting an appropriate buffer pH to accentuate charge differences between the proteins of interest. Complete separation of mg quantities of bovine serum albumin and ovalbumin was achieved within 40 min

Presented at the Second Annual Meeting of the Australian Electrophoresis Society, Sydney, April 29–30, 1995.

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Thermostable variants of subtilisin selected by temperature‐gradient gel electrophoresis
Authors: Sättler Andrea , Kanka Susanne , Maurer Karl‐Heinz , Riesner Detlev
Published: May 2005

Region‐specific random mutagenesis in the weak calcium binding site of subtilisin Carlsberg and subsequent screening for variants with enhanced heat stability revealed two variants, which showed significantly enhanced residual activity at 68°C, 0.1 mM CaCl2, pH 8.0. Preselected variants have been studied by temperature‐gradient gel electrophoresis (TGGE) and were found to be stabilized due to different effects. Whereas the point mutation (Ser188Pro) mainly enhanced autoproteolytic stability of subtilisin, the double mutation (Ser188Pro; Ala194Glu) additionally increased the apparent Tm‐value of the molecule for 2–3°C under a variety of conditions. It was possible to differentiate between the effects of autoproteolysis and structural unfolding to a certain degree by TGGE and to show the complex influence of changed calcium affinity on thermal stability for the double variant.
Determination of the C‐terminal form of an anemone toxin using capillary electrophoresis and mass spectrometry
Authors: Forest Eric , Pétillot Yves , Gagnon Jean , Cotton Joël , Vita Claudio
Published: May 2005

To identify the form of the C‐terminal amino acid of a sea anemone toxin, the native protein was compared with two synthetic proteins comprising the same sequence and a free or an amide C‐terminal form. Using electrospray ionization‐mass spectrometry, capillary electrophoresis and the coupling of both techniques, we assigned the C‐terminus of the native protein to be in the free carboxyl form.
Deterministic model of DNA gel electrophoresis in strong electric fields
Authors: Lee Namkyoung , Obukhov Sergei , Rubinstein Michael
Published: May 2005

We present a new model for the motion of a megabase‐long DNA molecule undergoing gel electrophoresis. We assume that the dynamics of large segments of DNA is almost deterministic and can be described by a set of simple mechanical equations. This allows the numerical study of gel electrophoresis of ultra‐high molecular weight DNA. A strong electric field forces DNA in a gel into a tree‐like structure with branches – loops of different sizes. We determined the loop‐size distribution function. This distribution has a power law form, confirming the hypothesis of the statistical self‐similarity of a moving polymer. We find periodic configuration changes in the motion of a circular polymer, with the average period proportional to the molecular weight. During the period, a polymer goes through three distinct phases: a simple V‐shape configuration, a growing tree, and a decaying tree. For a linear polymer this periodicity is much less pronounced because of additional perturbations to the dynamics caused by free ends. A circular polymer stays in a simple V‐shaped configuration about 30% of the time, independent of molecular weight (10% for a linear polymer).
Polyacrylamide solutions for DNA sequencing by capillary electrophoresis: Mesh sizes, separation and dispersion
Authors: Wu Chunhung , Quesada Mark A. , Schneider Dieter K. , Farinato Raymond , Studier F. William , Chu Benjamin
Published: May 2005

Two preparations of linear polyacrylamide with average molecular weights of 0.37 million and 1.14 million Da, and a deuterated preparation with an average molecular weight of 1.71 million Da, were used to study the effects of molecular weight, polydispersity, and concentration on the mesh size of entangled polymers in a DNA sequencing buffer solution and their ability to resolve DNA sequencing reactions by capillary electrophoresis. The polyacrylamide concentrations were above the overlap threshold C*, the concentration above which an entangled polymer network is expected to form. Small angle neutron scattering experiments showed that between 1% and 8% polyacrylamide, the mesh size (ζ) can be expressed by the relation ζ = 2.09C−0.76, where ζ is in Å and C is the polymer concentration in g/mL. The mesh size depended only on the concentration and was independent of the average molecular weight of the polyacrylamide. Consistent with this result, electrophoretic mobilities of DNA moving through the polymer network depended almost entirely on the polyacrylamide concentration and not on its molecular weight or polydispersity. Although separation was little affected, band sharpness persisted to longer DNAs when the polymer network contained a higher fraction of larger polyacrylamide molecuels. We postulate a dispersive effect that depends on the size of the DNA and the resiliency of the polymer network. This interpretation provides a rationale for optimizing the design of polymer solutions to sieve DNA for sequencing by capillary electrophoresis.
On the solvent motion in electrophoretic systems
Authors: Ermakov Sergey V. , Zhukov Michael Y. , Righetti Pier Giorgio
Published: May 2005

A corrected model describing transport processes for multicomponent mixtures in electric field is proposed. This model is more consistent compared to the other models of this sort used for simulations of electrophoresis in the case of concentrated solutions. The main idea underlying the model is in accounting for the motion of a solvent. Usually the concentrations of solutes are considered small compared to that of a solvent and the equation describing its motion is not considered. This automatically leads to the violation of momentum balance and, hence, to the defects of the model itself. In the model presented in the paper by means of redefining the mass fluxes the balance of momentum is satisfied automatically while the equations governing the evolution of the system look more symmetric and simple. This model allows us to discover some fine effects in evolution of the mixture and clarify the essence of some conservation laws (conservation of the Kohlrausch function) arising in the simplified mathematical models describing electromigration phenomena.
Northern blot analysis of simple repetitive sequence transcription in plants
Authors: Gortner Götz , Pfenninger Markus , Kahl Günter , Weising Kurt
Published: May 2005

The presence of simple repetitive sequence motifs in RNA from various plant species was probed by Northern blot analysis. Hybridization of total, poly(A)+‐ and poly(A)−‐RNA with microsatellite‐complementary oligonucleotide probes revealed distinct bands with most but not all probe/species combinations, demonstrating the presence of di‐, tri‐ and tetranucleotide repeat motifs in plant transcripts. Only trinucleotide repeat‐derived hybridization signals were found to be enriched in the poly(A)+‐fraction. The quality of Northern blot signals proved to be highly dependent on hybridization stringency. Thus, under the stringency conditions usually applied for oligonucleotide hybridization, some probes [(GT)8, (CAC)5, (TCC)5, and (CCTA)4] cross‐hybridized to bands corresponding in size to 18S and/or 26S rRNA. Cross‐hybridization to rRNA was significantly reduced at higher stringencies. These results stress the importance of carefully adjusting the hybridization conditions in Northern blot analysis of simple sequence transcripts.
(GTG)5 allows the distinction between different isolates of the nematode Caenorhabditis elegans
Authors: Benecke Mark , Epplen Jörg T. , Schierenberg Einhard
Published: May 2005

We have not been able to distinguish different isolates from the nematode Caenorhabditis elegans by morphological means. However, they differ on the molecular level and strains from several geographic regions can be identified with the help of “genetic fingerprints” using the oligonucleotide probe (GTG)5.
Determination of the kinetic parameters of adenosine deaminase by electrophoretically mediated microanalysis
Authors: Saevels Jan , Van Schepdael Ann , Hoogmartens Jos
Published: May 2005

The possibility of determining the Michaelis constant of the irreversible deamination of adenosine to inosine by adenosine deaminase, using capillary electrophoresis, was investigated. This paper describes the use of electrophoretically mediated microanalysis (EMMA) as the technique for carrying out the assay. Initial reaction velocities of the enzymatic reaction were estimated from the peak area of inosine, and the Michaelis constant was calculated according to the Lineweaver‐Burk equation. The result (Km = 5.3 × 10−5 M ± 8 × 10−6 M) was consistent with previously reported values. Using the present method, a total amount of as few as 1.2 fmole of enzyme and 9.2 ng of substrate were injected in the capillary for the construction of a Michaelis Menten curve (seven concentrations of substrate, each concentration analyzed in triplicate), which is far smaller than the quantities required in conventional methods.