bluuebunny/crossref_metadata_2025_split huggingface dataset:
Separating field strength, temperature, and pulsing effects in pulsed field electrophoresis
Authors: Mouradian Stéphane , Brumley Robert L. , Smith Lloyd M. 
Published: May 2005

AbstractThe utility of pulsed field electrophoresis for DNA sequencing is investigated. Previous studies have indicated a beneficial retardation of sequencing fragments when pulsed fields are employed. The interpretation of these results is complicated, however, by concomitant variations in electric field strength and/or temperature. Methods are presented here permitting discrimination of such mobility effects due to pulsing, field strength, and temperature. It is demonstrated that under the conditions employed here, observed mobility effects are due to electric field variations rather than pulsing. These conditions thus correspond to the low frequency/small molecule limit. The effect of temperature is estimated from the steady state solution to the heat conduction equation under appropriate boundary conditions. No temperature effect upon mobility is operative in the thin gel system employed, due to the high efficiency of heat transfer. However, it is shown that in conventional gel systems large temperature‐related mobility effects can occur. These methods for dissecting and understanding mobility effects in pulsed field electrophoresis are expected to be of general utility.
A large scale preparative gel electrophoresis separation of α1 and α2 subunits of the voltage‐gated Ca2+ channel from rabbit skeletal muscle
Authors: Nyormoi Okot , Schneider Toni , Smith Robert G. 
Published: May 2005

AbstractA large‐scale preparative gel electrophoresis method effectively separates individual voltage‐gated calcium channel (VGCC) subunits. with high resolution, starting with up to 4 mg of rabbit skeletal muscle L‐type VGCC complex. Using this method, we separated α1 and α2 subunits of rabbit skeletal muscle VGCC with a high efficiency and with protein recoveries of 83%. The separated α1 and α2 subunits eluted from the gel in a 1:1 molar ratio. The method should be applicable for separating the other VGCC subunits or subunits of other protein complexes.
Protein study of T and B acute lymphoblastic leukemia cell lines
Authors: Mohammad Ramzi M. , Vistisen Kerry , Al‐Katib Ayad 
Published: May 2005

AbstractTwo‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was used to identify cellular proteins in T and B acute lymphoblastic leukemia (ALL) cell lines. Five lines, REH and BALL‐1 of B‐cell lineage, CCRF‐CEM and HPB‐ALL of T‐cell lineage, and a normal Epstein‐Barr virus (EBV)‐transformed line of B‐origin (SKLN1) were studied. The lines were immunophenotyped using flow cytometry and lineage associated monoclonal antibodies. Whole cell lysates of the cell lines were subjected to 2‐D PAGE analyses. 2‐D gels were analyzed with an image scanning computer and the qualitative as well as quantitative differences of the protein patterns were studied. Despite the great similarities in the patterns of the B‐ and T‐gels, three proteins were unique to B‐cell lines, while eight were unique to T‐cell lines. Using cell lines is the first step toward identifying potential markers in ALL and can provide important information regarding the human ALL databases. Whether these proteins are definite markers for B‐ or T‐ALL or are unique to the cell lines studied needs further exploration.
Diode array detection in capillary electrophoresis
Authors: Heiger David N. , Kaltenbach Patrick , Sievert Hans‐Jürgen P. 
Published: May 2005

AbstractDiode array detection (DAD) in capillary electrophoresis (CE) offers similar advantages over single‐wavelength detection as it does in high performance liquid chromatography (HPLC). Thus, confirmation of compound identity and establishment of peak purity are critical issues in CE, necessitating sensitive and specific detection. With an optimized optical system, DAD yields sensitivity comparable to that of single or variable wavelength detectors. Sensitivity can be further improved three to five times by use of expanded pathlength capillaries employing the so‐called bubble cell. Optimization of optical design, as well as maintenance of spectral fidelity, will be discussed in this work. A variety of applications of CE, and specifically of micellar electrokinetic capillary chromatography (MEKC), with emphasis on quantitative analysis, sensitivity, linearity, spectral identification, and peak purity verification will be presented. The use of spectral information for peak tracking in MEKC method development and for the assessment of purity of electrodistorted peaks will also be illustrated.
Optimized separation of seven corticosteroids by micellar electrokinetic chromatography
Authors: Jumppanen Juho H. , Wiedmer Susanne K. , Sirén Heli , Riekkola Marja‐Liisa , Haario Heikki 
Published: May 2005

AbstractThe corticosteroids studied can be effectively separated by employing micellar electrokinetic capillary chromatography (MECC). The effect of pH, borate concentration and the sodium dodecyl sulfate (SDS) concentration on both the resolution and the selectivity was studied under 15 different experimental conditions. The experimental design was similar to the central composite design approach. Empirical quadratic regression models were derived for analyte migration time, band broadening and analyte velocity. Satisfactory regression fits and coefficients of determination for prediction were obtained with cross‐validated models. The models for analyte migration time and analyte velocity were in good agreement with theory. Modeling of the band broadening seemed to be somewhat more complicated. Optimum conditions for resolution and selectivity were different. This is due to the fact that selectivity studies ignore the electroosmotic and band broadening properties of different electrolyte solutions. However, the study of the selectivity yielded good information about the suitability of the electrolyte systems for the particular separation problem. Although a high solubilizing power of SDS caused the corticosteroids to partition strongly into the SDS micelles, a good separation could be achieved at low SDS concentrations.
Chiral separation of diniconazole, uniconazole and structurally related compounds by cyclodextrin‐modified micellar electrokinetic chromatography
Authors: Furuta Ritsuko , Doi Tadashi 
Published: May 2005

AbstractEnantiomeric separation of the structural analogs, diniconazole and uniconazole, can be achieved by cyclodextrin (CD)‐modified micellar electrokinetic chromatography (MEKC). The elution behavior of other structurally related compounds was therefore investigated and compared with that in high‐performance liquid chromatography (HPLC) using a CD‐bonded stationary phase. From the results, it was concluded that the benzene ring of the solute plays an important role in formation of CD complexes and chiral recognition in MEKC as well as in HPLC, although the enantioselectivity with the two methods was different. Nuclear magnetic resonance (NMR) data revealed different CD effects on the solute between the MEKC and HPLC models.
Nonaqueous packed capillary electrokinetic chromatographic separations of large polycyclic aromatic hydrocarbons and fullerenes
Authors: Whitaker Kylen W. , Sepaniak Michael J. 
Published: May 2005

AbstractPolycyclic aromatic hydrocarbons (PAH) and a fullerene mixture (C60/C70) were separated with capillary columns (50 μm ID) packed with a reversedphase packing (octadecylsilica, 3 μm diameter) using electrokinetic pumping. Nonaqueous (acetonitrile modified with methylene chloride or tetrahydrofuran, THF) mobile phases were used for these experiments. The effects of mobile phase composition on such factors as electroosmotic flow, plate height, and capacity factor (K′) are reported. The less polar solvents methylene chloride and THF produced predictable reductions in K′ when used to modify an acetonitrile mobile phase. Large amounts of the less polar modifiers (50% v/v) also resulted in a fourfold decrease in flow rate. This meant that even with a decrease in K′, the retention time increased. Nonaqueous capillary electrokinetic chromatographic (CEC) separations gave efficiencies as high as 160000 plates/m. Use of nonaqueous mobile phases provided small currents which in turn diminished the role of heating effects on efficiency. The nonaqueous system also provided greater solubility for the hydrophobic solutes. A Van Deemter plot for an acetonitrile mobile phase was obtained that exhibited expected trends in plate height with flow rate and K′. Solvent rinses with water and THF are shown to have only small effects on retention and flow rate.
Simulation of reduced band broadening during single‐stranded DNA pulsed field electrophoresis in polyacrylamide gels
Authors: Mayer Pascal , Slater Gary W. , Drouin Guy 
Published: May 2005

AbstractUsing a computer simulation algorithm based on the reptation model, we investigated the effects of pulsed field gel electrophoresis on the separation of single‐stranded DNA molecules in denaturing polyacrylamide gels. Pulsed fields that combine two different field intensities were found to affect the orientation of the reptation tube as well as the electrophoretic velocity, the rate of band broadening, the plate height and the molecular length for which the minimum of mobility was found, in agreement with available experimental results. Due to “memory” effects, pulses alternating between the forward and backward directions can reduce the diffusion constant by many orders of magnitude. Pulses of identical polarity but different intensities reduce the diffusion because stretches of less‐oriented DNA segments are conserved during the migration. We suggest that, for DNA sequencing, pulsed fields of fixed polarity should be used to reduce band broadening and not to overcome band inversion or suppress molecular orientation. We conclude that, using a low intensity, constant electric field will lead to smaller band widths than any pulsed field regime, in a similar length of experimental time, but with less complications.
Protein identifications for a Saccharomyces cerevisiae protein database
Authors: Garrels James I. , Futcher Bruce , Kobayashi Ryuji , Latter Gerald I. , Schwender Brian , Volpe Tom , Warner Jonathan R. , McLaughlin Calvin S. 
Published: May 2005

AbstractThe rapid progress in understanding the genes of the yeast Saccharomyces cerevisiae can be supplemented by two‐dimensional (2‐D) gel studies to understand global patterns of protein synthesis, protein modification, and protein degradation. The first step in building a protein database for yeast is to identify many of the spots on 2‐D gels. We are using protein sequencing, overexpression of genes on high‐copy number plasmids, and amino acid analysis to identify the proteins from 2‐D gels of yeast. The amino acid analysis technique involves labeling yeast samples with different amino acids and using quantitative image analysis to determine the relative amino acid abundances. The observed amino acid abundances are then searched against the current database of 2600 known yeast protein sequences. At present about 90 proteins onour yeast maps have been identified, and the number is rising rapidly. With many known proteins on the map, it will soon be possible to use 2‐D gel analysis to study regulatory pathways in normal and mutant yeast, with knowledge of many the protein products that respond to each genetic or environmental manipulation.
Capillary zone electrophoresis with indirect photometric detection in the visible range
Authors: Malá Zdena , Vespalec Radim , Boček Petr 
Published: May 2005

AbstractSensitivity and applicability of commonly used indirect photometric detection in the UV region can be adversely affected by the absorption of the UV light by detected solutes and/or by matrix constituents migrating with them. If the visible light is exploited instead of the UV light, both these disadvantages may be for the most part eliminated, and, moreover, the indirect photometric detection will extend its versatility and sensitivity. Prospects as well as main problems of the approach were tested using inorganic ions as analytes. If organic dyes with a molar light absorption coefficient of the order of 106 mol−1 L m−1 was selected as the light‐absorbing constituents of the background electrolyte, detection sensitivity comparable with the best reported results was reached for anions; for cations, the detection limit was even lower by two orders of magnitude.
Size‐dependent separation of proteins in the presence of sodium dodecyl sulfate and dextran in capillary electrophoresis: Effect of molecular weight of dextran
Authors: Karim Mohammed Rezaul , Janson Jan‐Christer , Takagi Toshio 
Published: May 2005

AbstractDextran solutions are widely used as sieving medium in protein analysis by capillary electrophoresis in the presence of sodium dodecyl sulfate. We studied the effect of dextran molecular weight on the separation efficiency using different dextran preparations with wide and very narrow molecular weight distributions, in the range between 1270 and 2 000 000. Migration times and band broadening of proteins were significantly affected by the molecular weight of dextran. Migration times of proteins decreased as molecular weights of the dextrans decreased. Satisfactory separation of all the proteins was possible with all dextrans except those with molecular weights of 70 000 and 23 800 where bovine serum albumin and phosphorylase b failed to be separated. Unexpectedly rapid separation of all the proteins with enhanced resolution could be observed using two dextrans with a narrow molecular weight distribution, with molecular weights of 1270 and 5220. Clearly the use of dextran with higher molecular weight is not the only way to achieve efficient sreparation of proteins. The separation‐mechanism in the presence of the low molecular weight dextrans remains to be made clear in a future study.
Analysis of tyrosinase gene mutations using direct automated infrared fluorescence DNA sequencing of amplified exons
Authors: Oetting William S. , Fryer James P. , Oofuji Yasuhisa , Middendorf Lyle R. , Brumbaugh John A. , Summers C. Gail , King Richard A. 
Published: May 2005

AbstractThe ability to correctly diagnose the molecular cause of genetic diseases is becoming increasingly important in medicine. This requires an efficient method for the analysis of the DNA sequence of specific genes and the detection of mutations in affected individuals. We report a method to determine the mutations responsible for tyrosinase related albinism (OCA1) using a combination of polymerase chain reaction‐single stranded conformational polymorphism (PCR‐SSCP) analysis and direct DNA cycle sequencing using fluorescently labeled oligonucleotides and an automated DNA sequencer based on infrared fluorescence technology. This method allows DNA from several individuals to be sequenced quickly and simultaneously so that the specific location of each mutation and the carrier status of family members can be determined.
Gel polymerization in detergents: Conversion efficiency of methylene blue vs. persulfate catalysis, as investigated by capillary zone electrophoresis
Authors: Caglio Silvia , Chiari Marcella , Righetti Pier Giorgio 
Published: May 2005

AbstractFour types of detergents are commonly used in biochemical analysis of proteins and polypeptides: neutral, e.g., Triton X‐100, Nonidet P‐40; anionic, typically sodium dodecyl sulfate (SDS); cationic, e.g., cetyltrimethylammonium bromide CTAB), and zwitterionic, e.g., sulfobetaine 3–12 and 3 [(3‐cholamido‐propyl) dimethyl‐ammonio]‐1‐propane sulfonate (CHAPS). These detergents are utilized not only in the protein solubilization step, but also in the polyacrylamide gel matrix in which subsequent electrophoretic separation is carried out. The conversion efficiency of monomers into the growing polymer, in the presence of the four types of detergents, was assessed by capillary zone electrophoresis (CZE) in a micellar system comprising 100 mM SDS, by extracting unreacted monomers from the gel phase and quantifying the peaks separated by CZE. Two different catalyst systems were evaluated: the standard persulfate‐N,N,N′,N′‐tetramethylethylenediamine (TEMED) couple and a mixture comprising methylene blue in presence of the redox couple sodium toluene sulfinate and diphenyliodonium chloride. In the chemically initiated system (persulfate), there was a strong inhibition of polymerization, decreasing in the following order: CTAB > sulfobetaine 3–12 > SDS > CHAPS > Triton X‐100 (e.g., in 10 mM CTAB 100% inhibition was experienced). On the contrary, in photopolymerization (as driven by methylene blue) good conversion of monomers into the growing polymer (> 95 %) was always obtained, independent of the nature of the detergent present in the polymerization step. It is suggested that acrylamide and N,N′‐methylenebisacrylamide (Bis) partition in the detergent micelles, thus being subtracted to equilibrium during polymerization: the order of inhibition in persulfate gels could then be proportional to the monomer partition coefficients. Conversely, the total lack of inhibition in methylene blue gels could be due to the extremely high performance of photocatalysis and/or to its partitioning also inside the detergent micelle.
Field programming to achieve uniform sensitivity for on‐line detection in electrophoresis
Authors: Shear Jason B. , Dadoo Rajeev , Zare Richard N. 
Published: May 2005

AbstractUnder constant applied electric field, an analyte band traverses an on‐line detection zone in an electrophoretic separation at a velocity inversely proportional to the detection time. Because analyte bands migrate with different velocities, the sensitivity of the detection system to different species can vary significantly. One approach for overcoming this problem is to change the applied electric field, E, as a function of the separation time, τ, so that the velocity of an analyte band when it passes through the detection zone is independent of the analyte's electrophoretic mobility. A possible solution is to make E(τ) grow exponentially, but this procedure causes band compression. The preferred solution is to make both E(τ) and the distance between the starting position of the sample and the detection zone increase linearly with separation time. This approach can be implemented with either slab‐gel or capillary electrophoresis.
Polymorphism of pepper (Capsicum annuum L.) seed proteins studied by two‐dimensional electrophoresis with immobilized pH gradients: Methodical and genetic aspects
Authors: Posch Anton , van den Berg Bartel M. , Duranton Claude , Görg Angelika 
Published: May 2005

AbstractA population of ten morphologically similar inbred lines of pepper (Capsicum annuum L.) has been investigated for polymorphism of seed proteins by two‐dimensional (2‐D) electrophoresis with immobilized pH gradients. To reveal as much variation as possible, both the water‐ and the urea/detergent‐soluble protein fraction were electrophoretically analyzed and subsequently visualized by silver staining. The 2‐D patterns were subjected to computer analysis to be able to establish genetic variation. A high number of the seed proteins were found to be variable as to presence/absence: these were 68 out of 184 reproducible water‐soluble proteins and 34 out of 419 reproducible urea/detergent‐soluble proteins. Comparison of the 2‐D data of the water‐soluble and the urea/detergent‐soluble proteins, which represent the biggest part of all extractable seed proteins, showed that both protein fractions have proteins in common, but the variable proteins found in both fractions were non‐identical. The difference of variability scored in both solubility fractions was discussed. Genetic distances between all pairs of inbred pepper lines were calculated and a genetic tree was constructed. A correlation analysis was carried out to correct for genetic linkage and for secondary modifications, to have a more proper estimate of genetic distances. In both cases the dendrograms showed two distinct genetic groups of five inbred lines. This electrophoretic study was done in order to utilize the genetic distance data in breeding for heterosis. The genetic distance data presented will be used to validate the assumption that there is a higher chance to achieve better hybrid performance when the genetic distance between the parents is as great as possible. From the fact that we found a high level of genetic variability within a population of ten morphologically similar inbred lines, it can be concluded that 2‐D electrophoresis can be efficiently applied in pepper breeding.
A qualitative and quantitative protein database approach identifies individual and groups of functionally related proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes: An overview of the functional changes associated with the transformed phenotype
Authors: Celis Julio E. , Olsen Eydfinnur 
Published: May 2005

AbstractA qualitative and quantitative two‐dimensional (2‐D) gel database approach has been used to identify individual and groups of proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes (K14). Five hundred and sixty [35S]methionine‐labeled proteins (462 isoelectric focusing, IEF; 98 nonequilibrium pH gradient electrophoresis, NEPHGE), out of the 3038 recorded in the master keratinocyte database, were excised from dry, silver‐stained gels of normal proliferating primary keratinocytes and K14 cells and the radioactivity was determined by liquid scintillation counting. Two hundred and thirty five proteins were found to be either up‐ (177) or down‐regulated (58) in the transformed cells by 50% or more, and of these, 115 corresponded to known proteins in the keratinocyte database (J. E. Celis et al., Electrophoresis 1993, 14, 1091–1198). The lowest abundancy acidic protein quantitated was present in about 60000 molecules per cell, assuming a value of 108 molecules per cell for total actin. The results identified individual, and groups of functionally related proteins that are differentially regulated in K14 keratinocytes and that play a role in a variety of cellular activities that include general metabolism, the cytoskeleton, DNA replication and cell proliferation, transcription and translation, protein folding, assembly, repair and turnover, membrane traffic, signal transduction, and differentiation. In addition, the results revealed several transformation sensitive proteins of unknown identity in the database as well as known proteins of yet undefined functions. Within the latter group, members of the S100 protein family – whose genes are clustered on human chromosome 1q21 – were among the highest down‐regulated proteins in K14 keratinocytes. Visual inspection of films exposed for different periods of time revealed only one new protein in the transformed K14 keratinocytes and this corresponded to keratin 18, a cytokeratin expressed mainly by simple epithelia. Besides providing with the first global overview of the functional changes associated with the transformed phenotype of human keratinocytes, the data strengthened previous evidence indicating that transformation results in the abnormal expression of normal genes rather than in the expression of new ones.