bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification of bioproducts by free‐flow zone electrophoresis: Choice of processing parameters
Authors: Roux‐De Balmann Hélène , Cerro Rose‐Marie , Sanchez Victor 
Published: May 2005

AbstractFree‐flow zone electrophoresis may be used to purify biological samples, due to differences in electrophoretic mobility, in absence of a matrix – most frequently a gel – thus enabling the biological integrity of even fragile molecules to be preserved. However, the process is more complicated than its principle suggests due to different transport phenomena interfering with electrophoretic migration, with the resultant separation depending both on separation effects and dispersive phenomena. The physical origin of the main effects involved was identified. Mathematical expressions were proposed to estimate the influence of the crescent effect and electrohydrodynamics on the process. In this paper, these equations are used to determine the minimum difference in electrophoretic mobility required for a separation to be achieved with respect to the processing parameters. A methodology is proposed which defines the conditions under which the difference in electrophoretic mobilities equals that calculated when considering the influence of dispersive phenomena. Optimized separations of the whey proteins lactoferrin and albumin, known to interact strongly, and the purification of a monoclonal antibody from a mouse ascitic fluid illustrate the approach.
Preparation of Golgi subfractions with free‐solution isotachophoresis: Analysis of sphingomyelin synthesis in Golgi subfractions from rat liver
Authors: Hofmeister Robert , Böttcher Alfred , Schmitz Gerd 
Published: May 2005

AbstractA new displacement electrophoresis technique, termed free‐solution isotachophoresis (FS‐ITP) was used for the analysis of sphingolipid metabolism in Golgi subfractions. The discontinuous electrolyte system enables tissue‐derived membrane vesicles to be separated and purified due to their polarity patterns in a mobility gradient. In this study total Golgi apparatus obtained from rat liver by discontinuous density gradient centrifugation was subfractionated by preparative FS‐ITP, yielding enzymatically active cis‐, medial‐, and trans‐Golgi subfractions. These membrane vesicles were assayed by the following established enzyme marker activities: NADH cytochrome c reductase (cis‐Golgi), NADP phosphatase (medial‐Golgi), and thiamine pyrophosphatase (trans‐Golgi). The activity of phosphatidylcholine:ceramide phosphocholine transferase, a sphingomyelin synthesizing enzyme, is attributed to the cis‐ and medial‐Golgi‐derived subfractions. Analysis of Golgi lipids revealed a decline in membranous ceramide along the cis‐ to trans‐Golgi polarity axis. Furthermore, significant amounts of newly synthesized sphingomyelin and diacylglycerol are transferred from the medial/cis‐ to the trans‐Golgi compartment. The FS‐ITP system is well suited for micropreparative experimental applications, as demonstrated by studies on phosphatidylcholin:exeramide phosphocholine transferase activity in Golgi membrane vesicles of rat liver obtained by FS‐ITP.
Separation and microgram‐scale isolation of sea urchin egg granules by electrophoresis in polyvinylpyrrolidone solution, using horizontal gel electrophoresis apparatus with fluorescence detector
Authors: Chen Nong , Radko Sergey P. , Chrambach Andreas 
Published: May 2005

AbstractA homogenate of sea urchin (Lytechinus pictus) eggs rich in exocytotic membrane vesicles (granules) was subjected to analytical and preparative electrophoresis in the commercial automated horizontal gel electrophoresis apparatus (HPGE‐1000, LabIntelligence, Belmont, CA) capable of intermittent scanning of the migration path, using buffered solutions of polyvinylpyrrolidone (PVP). The nonfluorescent granules were detected by “fluorescence reduction”, i.e., a decrease of fluorescence intensity due to the absorbance and/or light scattering properties of the particle. Granules migrated at linear migration rates in buffers ranging from 0 to 2.5% PVP. Two bands were observed and optimally separated in 1.5% PVP solution. As shown by sodium dodecyl sulfate (SDS)‐polypeptide patterns, the material recovered from the bands was qualitatively indistinguishable from the two major fractions A and C of granules previously separated by free‐flow electrophoresis in the absence of polymer. Ferguson plot analysis failed to provide the sizes of the granules in view of the narrow PVP concentration range available for mobility measurement and the unavailability of chemically homogeneous size standards.
Surface modification of superparamagnetic nanoparticles (Ferrofluid) studied with particle electrophoresis: Application to the specific targeting of cells
Authors: Sestier Claude , Da‐Silva Maria Fatima , Sabolovic Domagoj , Roger Jackie , Pons Jean Noël 
Published: May 2005

AbstractColloidal aqueous suspension of superparamagnetic nanoparticles (9 nm in diameter) composed of maghemite (γ Fe2O3) and forming an ionic ferrofluid in aqueous solution are covalently coupled with lectins, enzymes or antibodies, using specific thiol chemistry. The surface charge modifications of nanoparticles, caused by ligand coupling, were monitored by measuring their electrophoretic mobilities using laser‐Doppler velocimetry. Particle electrophoretic mobility (PEM) changes are shown to correlate well with the amount of ligand fixed on the particles, as probed by its biological activity. The PEM method provides a useful tool to optimize ligand immobilization at the surface of nanoparticles, and may be advantageous when biological activity measurements are not convenient.
Cell electrophoretic mobility and glycerol lysis of human erythrocytes in various diseases
Authors: Hashimoto Nobuya , Fujita Shuzo , Yokoyama Toru , Ozawa Yohinori , Kingetsu Isamu , Kurosaka Daitaro , Sabolovic Domagoje , Schuett Wolfgang 
Published: May 2005

AbstractUsing an automated cell electrophoresis system equipped with an image processor, we studied electrophoretic mobilites of erythrocytes of healthy donors and of patients suffering from systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), hypergammaglobulinemia and diabetes mellitus (DM). On average, erythrocytes from SLE patients showed mean electrophoretic mobilities (EPM) which were significantly lower (p < 0.005) than the EPM of red blood cells of normal donors. Evaluation of mean EPM and standard deviations revealed that three groups of SLE patients could be distinguished regarding the electrophoretic behavior of their erythrocytes. Some patients had red blood cells with normal EPM, others had erythrocytes with significantly reduced EPM, and a third group appeared to have both kinds of erythrocytes. In addition, erythrocytes of various SLE patients showed enhanced resistance to lysis by glycerol and their membranes contained less quantities of band 3 proteins.
Experimental study on temperature distribution within a wide‐gap continuous free‐flow electrophoresis chamber
Authors: Wang Haibo , Zhang Zhiyuan , Jiang Yuanda , Wu Hanji 
Published: May 2005

AbstractThe temperature distribution within a wide‐gap (3 mm) continuous free‐flow electrophoresis (CFFE) chamber was investigated by direct measurements. The walls of the chambers consisted of Plexiglas and thermocouples were used as temperature sensors. The study of different operating and heat dissipating conditions revealed that lateral temperature gradients arise in CFFE chambers in addition to an axial increase in temperature of 0.25°C per Watt of electric power and per minute of residence time in the absence of cooling.
Towards predicting mobility and resolution in polymeric media: Some first steps
Authors: Chrambach Andreas , Radko Sergey P. 
Published: May 2005

AbstractParticle size‐dependent retardation (“molecular sieving”) in electrophoresis can be achieved in polymer solutions or gels. In the semidilute concentration range of polymer solutions, mobility of “rigid, spherical” particles can be predicted from their size (in the size range less than 20 nm radius), the screening length specific for the particular polymer and a constant that can be experimentally determined for a polymer. That constant could be universal for all hydrophilic uncharged polymers. Band spreading in polymer solutions is constant over a range of particle sizes and polymer concentrations and increases beyond that range. Presumably, the critical division between the two ranges occurs when the diameter of the particle exceeds the screening length of the polymer network. Resolution, defined as separation divided by the sum of bandwidths, can thus be predicted for a limited particle size and polymer concentration range. In gels, resolution can be estimated from the slopes and free mobility intercepts of the Ferguson plot. However, the previous computation of resolution needs to be revised in view of the fact that band spreading in gels proceeds in proportion to time, presumably through interaction with the polymer, and not as a function of the square root of time as would be the case if band spreading resulted from diffusion.
Creutzfeldt‐Jakob disease: Clinical features, epidemiology and tests
Authors: Knight Richard 
Published: May 2005

AbstractCreutzfeldt‐Jakob disease (CJD) is now recognized to occur in four main forms: classical sporadic, genetic, iatrogenic and new variant. These are briefly described. The evidence that new variant disease is due to bovine spongiform encephalopathy (BSE) is reviewed. In CJD, disease and infectivity are associated with the accumulation of an abnormal form of a normal cellular protein. Protein analysis and the study of the gene which encodes for this protein have therefore played an important role in understanding CJD. Diagnostic tests for CJD are discussed with particular reference to the detection of the 14‐3‐3 protein in the cerebrospinal fluid (CSF).
Correlation of plasma protein separation patterns obtained by two‐dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis
Authors: Manabe Takashi , Miyamoto Hiromitsu , Oota Hideto , Toyota Yosuke 
Published: May 2005

AbstractWe used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)‐filled capillary electrophoresis with SDS (SDS‐CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2‐D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.
Membrane‐mediated sample loading for automated DNA sequencing
Authors: Cassel Sallie M. , Guttman András 
Published: May 2005

AbstractA new and improved sample loading method for DNA sequencing gels using oligo/polynucleotide binding membranes is described. The labeled DNA sequencing fragments were spotted onto a membrane sample loader, which was then placed in contact with the precast polyacrylamide separation gel. In this way, the time‐consuming sample well loading by the tedious pipetting procedure was avoided. The spotted DNA fragments remain immobilized on the membrane until the separation process is initiated by the application of the electric field (an active DNA release mechanism). This novel technique enables sample loading outside of the separation platform, thereby allowing full utilization of various automated sample preparation and liquid handling (robotics) systems, resulting in “real” automated DNA sequencing. The loaded membranes can be stored for more than 24 hours for later use.
Nonradioactive Northern blotting with biotinylated and digoxigenin‐labeled RNA probes
Authors: Meltzer Jon C. , Sanders Veronica , Grimm Paul C. , Chiasson Nathalie , Hoeltke Hans‐Joachim , Garrett Karryn L. , Greenberg Arnold H. , Nance Dwight M. 
Published: May 2005

AbstractThe application of nonradioactive RNA probes for Northern blotting offers the advantage of a rapid turn‐around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)‐labeled RNA probes from nylon membranes. One protocol utilizes a phosphate‐buffered formamide stripping solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripping solution. This latter method was developed by Ambion Inc. and is called Strip‐EZ™. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin‐labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called ChemLink™, that quickly and conveniently labels RNA for use in Northern blotting.
A 33 bp minisatellite repeat upstream of the ‘mutated in colon cancer’ gene at chromosome 5q21
Authors: Bugert Peter , Kenck Christiane , Kovacs Gyula 
Published: May 2005

AbstractWe have found a 33 bp minisatellite repeat in the 5′‐flanking region of the mutated in colon cancer (MCC) gene at chromosome 5q21. Southern blot experiments demonstrated the locus specificity of the repeat. The number of repeat units varied between 5 and 11 with a heterozygosity of 0.56. The sequence 5′‐AGG AGT GTG AAT GGG GCA TAG TGA ATG AGG GGA‐3′ of the repeat units does not match the consensus sequence of chi‐related minisatellites. The minisatellite is not expressed as part of a gene transcription unit. However, it can be used as a tool for the detection of allelic changes at chromosome 5q21 on standard agarose gels.
Ultrafast DNA analysis by capillary electrophoresis/laser‐induced fluorescence detection
Authors: Müller Odilo , Minarik Marek , Foret Frantisek 
Published: May 2005

AbstractThe limits of ultrafast DNA analysis by CE were determined by investigating the influence of the effective capillary length and the electric field strength on the analysis time for a given peak resolution (10 bp). In accordance with theory, the use of a fast ramp power supply for narrow plug electrokinetic injection was found to be essential to minimize the extra column effects on peak dispersion. Two major column dispersion factors, logitudinal diffusion and thermal dispersion, were determined experimentally, as well as the influence of the electric field strength on the electrophoretic mobilities and diffusion coefficients of DNA. It was found that higher field strengths can be applied with lower thermal dispersion than predicted by classical CE models. This was attributed to the faster mass transport in the radial direction due to field‐induced DNA orientation. Short capillaries (∼ 3–7 cm effective length) and moderate to high electric field strengths (∼ 600–800 V/cm) were used to perform a series of fast DNA separations. The dsDNA fragment standards ϕX174/HaeIII and pBR322/HaeIII were separated within 30 s. The possibility for fast mutation detection was demonstrated using constant denaturant capillary electrophoresis (CDCE) for the analysis of a single base mutation in mitochondrial DNA in 72 s. The potential for fast DNA sequencing was illustrated by separating 300 ssDNA fragments within 180 s.
The combined effect of acetonitrile and urea on the separation of polycyclic aromatic hydrocarbons using sodium dioctyl sulfosuccinate in electrokinetic chromatography
Authors: Luong John H. T. 
Published: May 2005

AbstractSodium dioctyl sulfosuccinate (DOSS) or sodium di2‐ethylhexyl sulfosuccinate, a relatively nontoxic and negatively charged surfactant, was selected and optimized for the capillary electrophoretic separation of the 16 Environmental Protection Agency (EPA) priority polycyclic aromatic hydrocarbons (PAHs). This pseudostationary phase displayed selectivities for PAHs which were somewhat similar to the C18 phase in reversed‐phase high performance liquid chromatography (HPLC). At high DOSS concentrations (> 30 mM), the hydrophobic interaction between DOSS and PAHs was pronounced and led to stronger retention of the latter. Consequently, acetonitrile was added to the running buffer to facilitate the elution of hydrophobic PAHs. In the absence of micelles, the separation mechanism was attributed to the solvophobic association of the PAH molecules with hydrophobic chains of the DOSS surfactant and there was a linear correlation between log k (retention factor) and the double bond number of the PAH. However, separations performed in an optimized buffer containing both DOSS and acetonitrile were not able to provide satisfactory performance. The separation of the 16 PAHs was then appreciably improved by adding urea to the running buffer to widen the separation window. At a high sample loading with UV detection, except for benzo[a]pyrene, benzo[b]fluoranthene and benzo[k]fluoranthene, all other PAHs were practically baseline‐resolved using an optimized running buffer containing 50 mM DOSS, 35% acetonitrile and 5 M urea in 8–10 mM borate, pH 9. Laser‐induced fluorescence permitted a very low sample loading and under such a running condition, a baseline resolution was obtained for these three most difficult PAHs. The addition of urea at this level, however, exhibited a noticeable quenching effect on the PAH fluorescent signal.
Proteins of rat serum: I. Establishing a reference two‐dimensional electrophoresis map by immunodetection and microbore high performance liquid chromatography‐electrospray mass spectrometry
Authors: Haynes Paul , Miller Ingrid , Aebersold Ruedi , Gemeiner Manfred , Eberini Ivano , Lovati Maria Rosa , Manzoni Cristina , Vignati Mara , Gianazza Elisabetta 
Published: May 2005

AbstractIn the present investigation, we have identified 56 major spots, or spot rows, corresponding to 22 proteins, in the 2‐DE pattern of adult male rats. This was done mainly by applying two complementary techniques, namely immunoblotting and high performance liquid chromatography‐mass spectrometry (HPLC‐MS) peptide mapping. Glycoproteins were characterized by affinity blotting with six lectins. We have also detailed how rat serum differs from human serum in two main respects: (i) relative abundance of individual proteins, which amounts in some cases to a complete absence in either sample, and (ii) varying molecular parameters for homologous proteins. It was thus possible to establish a first‐generation reference map of rat serum proteins, which can be accessed through http://weber.u.washington.edu/∼ruedilab/aebersold.html. We hope the present database will be a useful reference for the evaluation of changes in serum protein distribution in the course of pharmacological and toxicological studies. The recognition of species‐specific proteins appears of special relevance in this respect.
Agarose gel structure using atomic force microscopy: Gel concentration and ionic strength effects
Authors: Maaloum Mounir , Pernodet Nadine , Tinland Bernard 
Published: May 2005

AbstractAgarose gels have been studied by atomic force microscopy (AFM). The experiments were especially designed to work in aqueous conditions, allowing direct observation of the “unperturbed” gel without invasive treatment. AFM images clearly show strong dependence of pore diameter and its distribution on ionic strength of the solvent. As the ionic strength increases, the distribution becomes broader and the position of its maximum shifts toward higher values. The evolution of the distribution curves indicates that gels become more homogenous with decreasing Tris‐borate‐EDTA (TBE) buffer concentration. An empirical law of the mean pore diameter as a function of the ionic strength is established. In agreement with our previous work we found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide pore diameter distribution narrows as the gel concentration increases.
Recent developments in preparative free flow isoelectric focusing
Authors: Weber Gerhard , Boček Petr 
Published: May 2005

AbstractContinuous flow electrophoresis (CFE) is a promising method for preparative fractionation of a variety of biological species, ranging from peptides and proteins to subcellular particles and cells. The high separation efficiency of FFE may be deteriorated by hydrodynamic distortion of zones due to the omnipresent parabolic laminar flow profile. We show in this paper that the detrimental hydrodynamic distortion of separated proteins zones can be reduced, with resultant enhancement of separation efficiency, by employing continuous isoelectric focusing in pH gradients as the actual working regime in an advanced instrumentation. Newly developed media for fast generation of narrow‐ or broad‐range pH gradients under CFE conditions are described. The separation efficiency of these pH gradients is comparable to that of the gradients formed with the aid of synthetic carrier ampholytes. The new media are defined mixtures of nontoxic chemicals, and thus they are compatible with the requirements of human medicine. Experimental data are given showing that the new media offer fractionation of isoforms of proteins, that they offer resolution of proteins differing in isoelectric point (pI) by less than 0.05 pH units, and that these media inhibit proteins precipitation in experiments with human serum proteins.
Finding a universal low‐viscosity polymer for DNA separation
Authors: Heller Christoph 
Published: May 2005

AbstractWe have investigated the viscosity of different commercially available polymers in solution and found that dextran has a low viscosity compared to other polymers of comparable molecular weight and resolving power. This makes it a potentially useful matrix for DNA separation in capillary electrophoresis, where either short time or low pressure are preferred for matrix replacement. We showed that dextran performs well for the separation of oligonucleotides and double‐stranded DNA fragments. Together with the well‐known application for protein separation, this makes dextran a universal polymer for the separation of biological macromolecules.
Proteome and proteomics: New technologies, new concepts, and new words
Authors: Anderson N. Leigh , Anderson Norman G. 
Published: May 2005

AbstractThe goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment. Quantitative analysis of protein expression data obtained by high‐throughput methods has led us to define the concept of “regulatory homology” and use it to begin to elucidate the basic structure of gene expression control in vivo. Such investigations lay the groundwork for construction of comprehensive databases of mechanisms (cataloguing possible biological outcomes), the next logical step after the soon to be completed cataloguing of genes and gene products. Mechanism databases provide a roadmap towards effective therapeutic intervention that is more direct than that offered by conventional genomics approaches.