bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conventional isoelectric focusing and immobilized pH gradients in ‘macroporous’ polyacrylamide gels
Authors: Wenisch Elisabeth , de Besi Patrizia , Righetti Pier Giorgio 
Published: May 2005

AbstractLateral aggregation in presence of a hydrophilic polymer (e.g. 10 kDa polyethylene glycol) in the gelling solution (Righetti et al., Electrophoresis 1992, 13, 587–595) is not inhibited by high ionic strength nor in the pH 4–10 interval. However, the bundles are disaggregated by glycerol (Tm at 20 %) and by ethylene glycol (Tm at 24.5 %) as well as by pH extremes (pH 3 and pH 11). Supercoiling is also strongly inhibited in a copolymer, formed by acrylamide an N,N‐dimethylacrylamide or N‐methylacrylamide. A level of 50 % uncoiling is obtained well before a 1:1 ratio, already at a level of 18 % N,N‐dimethylacrylamide. All the above data strongly suggest that the nascent chains are held together in bundles by hydrogen bonds prior to the cross‐linking event, instead of having a random orientation and distribution in the solvent. However, it is not possible to distinguish between H‐bonds oriented perpendicular to the chain axis vs. H‐bonds occurring within a single polymer filament, and the two types of H‐bonds probably coexist. Macroporous gels perform well in steady‐state electrophoretic techniques, such as conventional isoelectric focusing and immobilized pH gradients, where a large‐pore structure is necessary for fast protein migration and for attainment of equilibrium conditions.
Dissociation and electrophoretic separation of dextranase and dextranase inhibitor from a tighly bound enzymeinhibitor complex of Streptococcus sobrinus
Authors: Wellington Janet E. , Shaw Janet M. , Walker Gwen J. 
Published: May 2005

AbstractEndodextranase was separated from dextranase inhibitor in culture filtrates of Streptococcus sobrinus by sodium dodecyl sulphate‐polylamide gel electrophoresis (SDS‐PAGE) in gel slabs containing blue dextran. Sample preparation included dissociation of the enzyme from its inhibitor by boiling for 1 min in SDS. During subsequent incubation of the gel, dextranase was located as clear bands on a blue background, and dextranase inhibitor appeared as blue zones on a clear background following incubation in dextranase solution. The enzyme and the inhibitor existed in multiple forms, and the range of molecular masses for dextranase (223–132 kDa) permitted an excellent separation from dextranase inhibitor (49–25 kDa). Although dextranase‐negative mutants, and wild type strains grown at low dilution rate in the chemostat, were devoid of free dextranase activity, the enzyme was easily located by analytical SDS‐PAGE. Likewise, analysis of filtrates from wild type strains, which contained no free inhibitor activity when growth occurred at high dilution rate, revealed dextranase inhibitor activity on the gels. The total production (free + combined) of dextranase and inhibitor by S. sobrinus was determined by dissociation of enzyme‐inhibitor complexes in concentrated cell‐free filtrates, their separation by preparative SDS‐PAGE and electroelution from the gels, followed by renaturation of protein activity. From a comparison of activity tests of free dextranase and free inhibitor in untreated filtrates with the results of similar tests on renatured electroeluates, the proportion of each constituent bound into a complex under each growth condition could be deduced.
Classification of mouse liver proteins by immobilized metal affinity chromatography and two‐dimensional electrophoresis
Authors: Jungblut Peter , Baumeister Hans , Klose Joachim 
Published: May 2005

AbstractMouse liver proteins were classified into metal‐binding and non‐binding proteins by combining immobilized metal ion affinity chromatography (IMAC) and twodimensional electrophoresis (2‐DE). The proteins were fractionated by three metal ions, Zn2+, Ni2+ and Cu2+, immobilized on iminodiacetic acid and then separated by 2‐DE. The total number of protein spots resolved by 2‐DE increased approximately twofold when the proteins were prefractionated by IMAC. By establishing 2‐DE standard patterns, 371 proteins were selected and then characterized according to their specificity in binding the three different metal ions. Only 48 proteins did not bind to any of the three metal ions investigated. Cu2+ was the most efficient ion in binding different proteins (310) compared to the other metals. Cu2+ bound to 42 proteins specifically and to 268 proteins unspecifically. Both Zn2+ and Ni2+ showed specific affinity only to four proteins.
Quantitative applications and validation of the resolution of enantiomers by capillary electrophoresis
Authors: Altria Kevin D. , Goodall David M. , Rogan Manus M. 
Published: May 2005

AbstractThe separation and quantitation of enantiomers is an important application of capillary electrophoresis. This paper reviews the performance and validation of capillary electrophoretic methods for this application. Limits of detection of < 0.1% are shown for determinations of minor enantiomer levels. Application areas include enantiomeric purity testing of pharmaceuticals and herbicides, reaction rate monitoring, stability testing, and clinical analysis. It is concluded that the number of quantitative application areas will undoubtedly expand as capillary electrophoresis becomes more widely established as an alternative and complement to high‐performance liquid chromatography.
A computer simulation accounting for dissimilar electrophoretic behavior between two similarly curved DNA fragments due to a difference in arc‐length
Authors: Wheeler David L. , Chrambach Andreas 
Published: May 2005

AbstractArc‐shaped bent DNA fragments of the same predicted planar curvature but differing in length by 20% were compared in regard to their mobilities in 3 to 10% polyacrylamide. The longer (155 bp) fragment is retarded far more severely than the shorter (124 bp) fragment. The effect of gel concentration in promoting the retardation is far more pronounced for the 155 bp than for the 124 bp fragment. Moreover, a temperature change from 25°C to 4°C does not substantially affect the gel concentration dependent mobility of the 124 bp fragment while it increases the retardation of the 155 bp fragment greatly. The strong increase in retardation brought about by a mere 20% increase in the length of the arc was accounted for by a simple computer simulation of gel electrophoresis which considered the rate of passage of arc‐shaped objects through a two‐dimensional array of disc‐shaped obstacles. Since the simulation relies exclusively on geometric factors, its success in predicting the behavior of the 124 and 155 bp DNA fragments suggests that geometric factors are largely responsible for their electrophoretic properties. The simulation can account for the strong temperature effect on the retardation of a model of the 155 bp DNA in polyacrylamide gels by showing that a decreased degree of random motion has a profound effect on the modeled 155 bp particle, but not on the modeled 124 bp DNA.
On the use of dimensionless parameters in acid‐base theory VII. The pH of solutions of salts of a strong acid and a weak base or vice versa
Authors: Rilbe Harry 
Published: May 2005

AbstractThe equation for the hydrogen ion activity in solutions of salts of a strong acid and a weak base, or vice versa, is of the thírd degree. Nevertheless, explicit equations for pH of such solutions can be formulated as linear functions of the logarithm of the salt concentration. The equations are valid within the major parts of pH and concentration intervals of experimental interest. The ranges of validity are given in terms of mathematically defined pH intervals. Close relations with pH of free base or free acid solutions of the same concentration as the salt are demonstrated. For instance, the pH difference between solutions of the salt and the free base or acid having unit activities is ± 7 pH units.
Immobilised ph gradient isoelectric focusing of wool proteins
Authors: Herbert Benjamin R. , Woods Joy L. 
Published: May 2005

AbstractConventional carrier ampholyte‐isoelectric focusing is unsuitable for separating wool and hair proteins. The inability to form stable narrow pH gradients at acidic pH values does not allow good resolution of the many acidic wool and hair proteins. To evaluate the usefulness of immobilised pH gradient‐isoelectric focusing (IPG‐IEF) for wool proteins, S‐amidomethyl wool proteins were analysed using IPG‐IEF. Over twenty bands were visible on a pH 4–7 IPG‐IEF gel. IPG‐IEF was combined with sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to give a two dimensional separation, and over 50 protein spots were observed. IPG‐IEF appears to be ideally suited to the separation of wool proteins. The resolution of two‐dimensional electrophoresis using IPG‐IEF was greater than previously obtained using acid or alkaline PAGE in the first dimension. Good reproducibility of spot position was observed.
Comparison of protein analysis between embryonic and extraembryonic tissues during the 11th day of gestation of the mouse
Authors: Baumgartner Bernhard G. , Murach Karl‐friedrich , Schlegel Elisabeth , Praxmayer Claudia , Illmensee Karl 
Published: May 2005

AbstractAt day 11 of gestation, embryos and their extraembryonic tissues were isolated from the uterus of Him OF1/SPF mice and incubated in Dulbecco's modified Eagle's medium (DMEM) containing L‐[35S]methionine. After 4 h of incubation, the embryos were dissected to obtain the heart, liver, limb buds, and brain. The latter was fragmented into the telencephalon, mesencephalon, and myelencephalon. These organs and the extraembryonic tissues such as chorion, yolk sac, and placenta were processed for two‐dimensional (2‐D) gel electrophoresis. About 1000 proteins with relative molecular weights (Mr) varying from 10 000 to 200 000 and isoelectric points ranging from 4 to 10 could be detected on these gels. The protein patterns of the various organs and tissues were analyzed for organ‐ and cell lineage‐specific protein spots. We detected subtle differences in the protein patterns of the three cerebral areas when compared to each other. In addition, we found protein spots characteristic for the entire brain. We also found several heart‐specific protein spots. Distinct protein synthesis was also detected in liver and limb buds. Several groups of protein spots seem to be differentially regulated in these organs. Substantial differences between the patterns of embryonic and extraembryonic tissues were observed. In addition, several clusters of protein spots of well‐defined molecular weight could be detected only in extraembryonic tissues. We propose that organ‐ and tissue‐specific differences in protein synthesis are linked to some of the morphogenetic and functional processes during mammalian embryogenesis. Identification of particular proteins will serve as a basis to search for the corresponding genes.
Application of micellar electrokinetic capillary chromatography for monitoring of hippuric and methylhippuric acid in human urine
Authors: Lee Kong‐Joo , Lee Jong Jin , Moon Dong Cheul 
Published: May 2005

AbstractThe factors affecting micellar electrokinetic capillary chromatographic separation of hippuric and o‐, m‐, p‐methylhippuric acid were investigated by changing the species of micelles, and adding urea to the micellar solution. The analysis of hippurates in human urine is demonstrated under optimum conditions using 20 mM phosphate buffer (pH 8.0) containing 100 mM dodecyltrimethylammonium bromide and 4 M urea at −22 kV applied voltage. This method proved suitable for the screening of hippurates in human urine following occupational exposure to toluene and xylene.
Electrophoretic analysis of coniferyl alcohol oxidase and related laccases
Authors: Udagama‐Randeniya Preethi , Savidge Rodney 
Published: May 2005

AbstractGradient gel electrophoretic methods enabled a distinction to be made between coniferyl alcohol oxidase (CAO) of lignifying cell walls and a pI ∼ 9 pine “laccase” recently implicated in lignification (Science 1993 260, 672). Following treatment of a partially purified protein mixture from developing xylem of Pinus strobus with 2‐[N‐morpholine]ethanesulfonic acid (MES) buffer, isoelectric focusing and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis indicated that CAO had been selectively precipitated by MES and thereby purified to electrophoretic homogeneity. Purified CAO was determined to be a cell‐wall‐bound glycoprotein (38% glycan), Mr 107 500, pI,7.6, pH and temperature optima 6.3 and 30°C, respectively. By graphite‐furnace atomic‐absorption analysis, CAO contained one copper atom per protein molecule. Proteins obtained from lignifying cambial derivatives of conifers (family Pinaceae) and from Rhus typhina bark were compared with CAO and the pI ∼ 9 pine “laccase” following electrophoresis and Western blotting. For Abies balsamea, Larix laricina, Picea rubens, Pinus banksiana, Pinus taeda, and R. typhina, the isoelectric points of oxidatively active bands were identical to those of purified CAO. In addition, for all species only the pI 7.6 band was immunoreactive with antibodies against periodate‐deglycosylated CAO.
Capillary zone electrophoresis of oligosaccharides derivatized with various aminonaphthalene sulfonic acids
Authors: Chiesa Claudia , O'Neill Roger A. 
Published: May 2005

AbstractMalto‐oligosaccharides were derivatized via their reducing ends with different aminonaphthalene mono‐, di‐ and trisulfonic acids by reductive amination. The derivatives were then separated by capillary zone eléctrophoresis in uncoated fused silica capillaries, using 50 mM triethylammonium phosphate buffer, pH 2.5, as running electrolyte. The effect of degree of charge on speed of analysis and resolution was studied for different aminonaphthalene mono‐, di‐ and trisulfonic acids. Under the conditions used, a higher degree of charge on the derivatives provided both faster analyses and higher resolution. Investigation of the electrophoretic behavior of derivatized oligosaccharides obtained from bovine pancreatic ribonuclease B gave insight into the possibility of applying such electrophoretic systems to the analysis of more complex carbohydrates. The resolution of positional isomers under the conditions described indicated that the high resolving power of this technique allows separations not strictly based on the effects of charge and mass of the analytes, but on structural characteristics as well. The relationship between electrophoretic mobility and molecular structure was investigated for the different derivatives.
Positional reproducibility of protein spots in two‐dimensional polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric focusing in the first dimension: An interlaboratory comparison
Authors: Corbett Joseph M. , Dunn Michael J. , Posch Anton , Görg Angelika 
Published: May 2005

AbstractAn intra‐ and interlaboratory comparison of positional reproducibility of protein spots in two‐dimensional electrophoresis using immobilised pH gradients (IPG) in the first dimension (IPG‐DALT) was made. Aliquots of two different samples, human cardiac and barley leaf proteins, were separated in two different laboratories (London and Munich), using 180 mm long IPG gel strips, pH 4–8, for the first dimension and homogeneous SDS‐PAGE gels (12% T) for the second dimension. Subsets of 340 (cardiac) and 200 (barley) well‐resolved spots distributed across the 2‐D gel patterns were selected for computer analysis (PDQUEST) of positional reproducibility. The IPG‐dimension was highly reproducible in each laboratory, with a mean standard deviation of about 1 mm for both types of sample. Interlaboratory comparisons revealed identical results for barley with a mean standard deviation along thex‐axis of about 1 mm, whereas the cardiac matchset showed slightly more variability (mean standard deviation ∼ 1.5 mm). Nevertheless, IPG‐DALT provides significantly improved reproducibility of spot positions compared to conventional isoelectric focusing with synthetic carrier ampholytes.
Gel electrophoresis of end‐labeled DNA II. Dynamics and detrapping in pulsed fields
Authors: Défontaines Anne‐Dominique , Viovy Jean‐Louis 
Published: May 2005

AbstractA theory for field‐inversion gel electrophoresis of a flexible polyelectrolyte bearing an uncharged bulky label at one end is described, and the evolution of the mobility with chain length, field strength, friction of the label, and the duration of the forward and reverse pulses is predicted. A new critical size, N detrap, is introduced, and its value calculated. It increases roughly linearly with the duration of the reverse pulses. Chains smaller than Ndetrap are detrapped by reverse pulses, and may have a high mobility, whereas chains larger than N detrap are not trapped, and have a very small mobility. This leads to an increase of the mobility (as compared with constant field) in a given range of sizes, and to a strong selectivity around N detrap Depending on the parameters, numerous other effects, including a secondary mobility plateau and band inversion, may appear. The corresponding regimes are predicted and discussed. All predictions are qualitatively consistent with available experimental data. We use them to suggest efficient conditions for the development of pulsed‐field trapping electrophoresis, a possible tool for improved DNA sequencing. In particular, we recommend using a ramping of pulse times, with a constant ratio of forward to reverse time in the range 3 to 5.
The human keratinocyte two‐dimensional protein database (update 1994): Towards an integrated approach to the study of cell proliferation, differentiation and skin diseases
Authors: Celis Julio E. , Rasmussen Hanne Holm , Olsen Eydfinnur , Madsen Peder , Leffers Henrik , Honoré Bent , Dejgaard Kurt , Gromov Paul , Vorum Henrik , Vassilev Alex , Baskin Yasemin , Liu Xiangdang , Celis Ariana , Basse Bodil , Lauridsen Jette B. , Ratz Gitte P. , Andersen Annette H. , Walbum Else , Kjærgaard Inge , Andersen Inger , Puype Magda , Van Damme Josef , Vandekerckhove Joël 
Published: May 2005

AbstractThe master two‐dimensional (2‐D) gel database of human keratinocytes currently lists 3087 cellular proteins (2168 isoelectric focusing, IEF; and 919 nonequilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to posttranslational modifications. 890 polypeptides have been identified (protein name, organelle components, etc.) using one or a combination of procedures that include (i) comigration with known human proteins, (ii) 2‐D gel immunoblotting using specific antibodies (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry and (v) vaccinia virus expression of full length cDNAs. These are listed both in alphabetical order and with increasing SSP number, together with their Mr, pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore, we list 239 microsequenced proteins recorded in the database. We also report a database of proteins recovered from the medium of noncultured, unfractionated keratinocytes. This database lists 398 polypeptides (309 IEF; 89 NEPHGE) of which 76 have been identified. The aim of the comprehensive databases is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and, ultimately, to pinpoint signaling pathways and components affected in various skin diseases, cancer included.
REF52 on global gel navigator: An internet‐accessible two‐dimensional gel electrophoresis database
Authors: Boutell Thomas , Garrels James I. , Franza B. Robert , Monardo Pat J. , Latter Gerald I. 
Published: May 2005

AbstractUntil recently, the REF52 2‐D gel database of experiments with rat cell lines was accessible only with special software. This database has now been made available to all investigators with access to the Internet, using the World Wide Web (WWW) technology. The package which delivers the database through the WWW has been named the Global Gel Navigator and can be used to explore the data by several methods, including the direct selection of proteins in the displayed gel using the mouse.
Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients
Authors: Gelfi Cecilia , Righetti Pier Giorgio , Cremonesi Laura , Ferrari Maurizio 
Published: May 2005

AbstractA new and fast method is described for detection of point mutations in polmerase‐chain reaction (PCR)‐amplified DNA, based on capillary electrophoresis in sieving liquid polymers in presence of temporal thermal gradients. The background electrolyte contains a constant amount of denaturing agent (e.g., 6 M urea) and the DNA fragments are injected in a constant‐temperature plateau below the melting temperature (Tm). After loading, a temperature ramp is activated (typically from 0.2 to 0.6°C/min, according to the melting profiles of the DNA duplexes under investigation) with resultant branching of homo‐ and heteroduplexes at different times along the migration path. In the case of individuals heterozygous for a point mutation, the expected four‐band pattern is obtained. The temperature gradient is not produced externally, via circulating coolant and a thermostat, but is generated internally by using a dedicated computer program able to calculate the precise inner temperature under given electric conditions. The method is applied to the identification of three point mutations located in exo 17b (R1066H, R 1066C, F1052V) and of two polyrmorphisms located in exon 14a (V868V, T854T) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
Consequences of a maximum existing in the dependence of separation selectivity on concentration of cyclodextrin added as chiral selector in capillary zone electrophoresis
Authors: Vespalec Radim , Fanali Salvatore , Boček Petr 
Published: May 2005

AbstractThe maximum in the dependence of the separation selectivity on the concentration of cyclodextrin may be utilized for the determination of the mean value of host‐guest interaction constants of the separated enantiomers. For the hosting of N‐t‐BOC‐DL‐tryptophan by β‐cyclodextrin, the mean value of these constants, found by drop line estimated from the cyclodextrin concentration corresponding at the maximum, is 350 L.mol−1. The separation at this concentration of cyclodextrin offers the highest resolution in the shortest separation time. It is shown that commonly used simple preliminary experiments, testing the capability of a cyclodextrin to resolve chiral compounds, and based on relatively high cyclodextrin concentrations, may easily lead to incorrect conclusions in cases of enantiomers strongly interacting with the cyclodextrin used.
Capillary zone electrophoresis for the rapid screening of banned drugs in sport
Authors: Gonzalez Esther , Laserna Jose J. 
Published: May 2005

AbstractCapillary zone electrophoresis was used to analyze some drugs illegally used in sports. The spectroscopic characteristics and electrophoresis migration parameters of diuretics, narcotics‐analgesics and β‐blockers were established. Analytical figures of merit including parameters on the precision of migration and limits of detection are discussed. Results for the separation of drugs of different doping families are discussed. An analysis of urine from a patient receiving daily dosis of atenolol is presented.