bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isoelectric focusing of apolipoproteins in immobilized pH gradients: Improved determination of apolipoprotein Ephenotypes
Authors: Baumstrak Manfred W. , Berg Aloys , Halle Martin , Keul Joseph 
Published: May 2005

AbstractA method for isoelectric focusing of apolipoprotein Ein an immobilized pH gradient with added carrier ampholytes has been developed. This method is an improvement over conventional isoelectric focusing of apolipoprotein E with respect to resolution reproducibility, and simplicity. Since monosialo isoforms are resolved from the not mally cofocusing asialo isoforms, unique patterns are obtained for all 6 common apolipoprotein E phenotypes. The method can also be applied to the screening of apolipoprotein A and C isoforms. Delipidated very low density lipoproteins (VLDL) have been used as the source of apolipoprotein E and C. Apolipoprotein A isoforms were focused directly from detergent‐treated serum. Immunodetection of apolipo protein E using capillary transfer was found to be compatible with the described method.
Isoelectric focusing in immobilized pH gradients with carrier ampholytes added for high‐resolution phenotyping of bovine β‐lactoglobulins: Characterization of a new genetic variant
Authors: Krause Ingolf , Buchberger Johann , Weiß Günther , Pflügler Monika , Klostermeyer Henning 
Published: May 2005

AbstractThe genetic variants of bovine β‐lactoglobulin (β‐lg) from the “Murnau‐Werdenfelser” breed were analyzed in three different isoelectric focusing (IEF) systems. While carrier ampholyte IEF with a pH gradient of 0.2 pH/cm did not resolve the new variant W from the B variant and IEF in immobilized pH gradients (IPG) with 0.1 pH/cm only partially resolved it, adequate separation was achieved with IPG‐IEF in a pH 5.25‐pH 5.7 gradient, in presence of 0.8 % w/v carrier ampholytes, both over a 10 and 17 cm separation distance. Apparent isoelectric points (pI's) and genetic frequencies (f) were as follows: β‐lg A, pI = 5.370,f = 0.364; β‐lg B, pI = 5.485,f = 0.480; β‐lg W, pI = 5.492,f = 0.076; and β‐lg D, pI = 5.610,f = 0.080. The small difference of Δ pI = 0.007 between β‐lg B and β‐lg W respectively, seems to originate from a “silent” substitution of neutral amino acid residues as compared to the larger Δ pI's of the other genetic variants of β‐lg, which result from substitution of charged amino acids.
Isoelectric focusing of alpha‐1 acid glycoprotein (orosomucoid) in immobilized pH‐gradients with 8 M urea: Detection of its desialylated variants using an alkaline phosphatase‐linked secondary antibody system
Authors: Eap Chin B. , Baumann Pierre 
Published: May 2005

AbstractA method allowing a clear separation of the different variants of desialylated alpha 1‐acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2 % v/v 2‐mercaptoethanol. Immunoblotting with two antibody‐steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1‐acid glycoprotein in a 20 μL plasma sample diluted 1:28 672. A one year old bloodstain, kept at room temperature, could easily be phenotyped.
Photocontrol of organelle and cell type specific changes in the polypeptide composition of Euglena and sorghum
Authors: Monroy Antonio F. , Gardiner William E. , Schwartzbach Steven D. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) gel electrophoresis has been used to follow changes in cell type specific and organelle localized polypeptides upon exposure of etiolated sorghum shoots and dark‐grown resting Euglena to light. Total protein extracted from isolated bundle sheath strands and mesophyll protoplasts was resolved by 2‐D gel electrophoresis. The cell type specific polypeptides were localized on the whole shoot 2‐D gel map in order to determine changes in the levels of these polypeptides upon light exposure. An image analyzer was used to analyze fluorographs of 2‐D gels of total Euglena protein pulse‐labeled with [35S]sulfate in the dark, immediately upon light exposure and 1, 4, 14, 24, 48 and 72 h after light exposure. The subset of polypeptides whose relative rats of synthesis changes more than threefold immediately upon light exposure was i dentified. The different patterns of changes in the rate of synthesis of this subset of polypeptides were followed.
Two‐dimensional gel analysis of yeast proteins: Application to the study of changes in the levels of major polypeptides of Saccharomyces cerevisiae depending on the fermentable or nonfermentable nature of the carbon source
Authors: Bataillé Nelly , Thoraval Didier , Boucherie Hélian 
Published: May 2005

AbstractTaking advantage of the recent identification of polypeptides of the carbor meta. bolism machinery on the yeast protein map [1], we applied two‐dimensional gel electrophoresis to a study of changes in protein composition of Saccharomyces cerevisiae depending on the fermentable or nonfermentable nature of the carbon source. The levels of the 250 most abundant polypeptides were compared. Thirty‐three were found to display markedly increased levels during growth on nonfermenable carbon sources. These 33 polypeptides include 11 mitochondrial polypeptides and polypeptides corresponding to alcohol dehydrogenase II, acetyl‐CoA synthetase, phosphoenol pyruvate kinase and hexokinase PI. Sixteen other polypeptides, in contrast, reached their higher levels during growth on fermentable corbon sources. Among these were identified the monomeric subunits of 6 giycoytic enzymes. Collectively the 33 polypeptides of the first class comprised over 30% of the total soluble proteins of cells grown on nonfermentable carbon source and 3 % during growth on fermentable carbon source. The protein fraction of the 16 polypeptides of the second ass corresponded to 10 % and 38 %, respectively. Together these results show that two‐dimensional gel electrophoresis, when coupled with the identification of polypeptides of the carbon metabolism apparatus, provides a valuable tool for approaching questions concerning carbon metabolism in S. cerevisiae.
Polypeptide changes induced by salt stress, water deficit, and osmotic stress in barley roots: A comparison using two‐dimensional gel electrophoresis
Authors: Hurkman William J. , Tanaka Charlene K. 
Published: May 2005

AbstractTwo‐dimensional polyacrylamide gel electrophoresis was used to analyze and compare the effects of short term treatments (24 h) of salt stress, water deficit (desiecation), and osmotic stress (polyethylene glycol and mannitol) on protein synthesis in roots of barley seedlings (Hordeum vulgare L. cv. CM 72). These comparisons were made to determine if the polypeptides of Mr 26 000 and 27 000 and pI of 6.3 and 6.5 that were observed previously to increase significantly with salt stress (Plant Physiol. 1987, 83 517–524) also increased with water deficit and osmotic stress. The polypeptide patterns for control‐and stress‐treated plants were qualitatively similar, but the net synthesis of a number of polypeptides was quantitatively altered by each of the stress treatments. Of the polypeptide changes induced by the stress treatments, many were unique to a specific stress. Other polypeptide changes were common between two or more of the stress treatments. Only one polypeptide change, a decrease, was common to all of the stress treatments. An important finding was that polypeptides that increased significantly in response to salt stress did not increase in response to water deficit or osmotic stress.
The use of a bivalent counter anion to control the effective mobility of the hydrogen ion constituent in cationic isotachophoresis
Authors: Sudor Ján , Stránský Zdeněk , Chmela Zdeněk , Boček Petr 
Published: May 2005

AbstractThe use of a bivalent counter anion for cationic isotachophoresis was studied both theoretically and experimentally. A mathematical model was proposed and solved to give the effective mobility of the hydrogen ion constituent, uH.H, in the respective electrolyte systems. The theoretical values agreed well with the experimental data in a series of electrolyte systems. The dependence of uH.H on such parameters as pH of the leading electrolyte, ionic mobilities of leading and counter ions, and dissociation constants was calculated and discussed. A bivalent counter anion will prove useful for the separation of low mobility cations and weak bases, as shown for the separation of substituted anilines in a succinate electrolyte system.
GESA – a two‐dimensional processing system using knowledge base techniques
Authors: Rowlands David G. , Flook Alan , Payne Peter I. , van Hoff Arthur , Niblett Tim , McKee Sean 
Published: May 2005

AbstractThe successful analysis of two‐dimensional (2‐D) polyacrylamide electrophoresis gels demands considerable experience and understanding of the protein system under investigation as well as knowledge of the separation technique itself. The present work concerns the development of a computer system for analysing 2‐D electrophoretic separations which incorporates concepts derived from artificial intelligence research such that non‐experts can use the technique as a diagnostic or identification tool. Automatic analysis of 2‐D gel separations has proved to be extremely difficult using statistical methods. Non‐reproducibility of gel separations is also difficult to overcome using automatic systems. However, the human eye is extremely good at recognising patterns in images, and human intervention in semi‐automatic computer systems can reduce the computational complexities of fully automatic systems. Moreover, the expertise and understanding of an “expert” is invaluable in reducing system complexity if it can be encapsulated satisfactorily in an expert system. The combination of user‐intervention in the computer system together with the encapsulation of expert knowledge characterises the present system. The domain within which the system has been developed is that of wheat grain storage proteins (gliadins) which exhibit polymorphism to such an extent that cultivars can be uniquely identified by their gliadin patterns. The system can be adapted to other domains where a range of polymorpic protein sub‐units exist. In its generalised form, the system can also be used for comparing more complex 2‐D gel electrophoretic separations.
The two radii of a charged particle
Authors: Michov Budin M. 
Published: May 2005

AbstractThe existence of two radii of each charged particle – a geometric and electrokinetic radii, is supposed. The mathematical relationship between them in the four possible combinations of an ion and its counterion is analyzed: (i) at equal geometric radii and, in absolute values, equal valencies; (ii) at equal geometric radii and, in absolute values, different valencies; (iii) at different geometric radii and, in absolute values, equal valencies; (iv) at different geometric radii and, in absolute values, different valencies. One of the equations worked out can be used to define the relationship between the geometric and electrokinetic radii of a polyion. All the equations are used in working out precise calculations.
Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries
Authors: Kilár Ferenc , Hjertén Stellan 
Published: May 2005

AbstractHuman serum transferrin is a mixture of isoforms (isoproteins) having different amounts of carbohydrates. Each isoform may exist in iron‐free and iron‐complexed molecular form. The genetic variations in different populations increase the number of combinations of the different forms of transferrin. To resolve the many components in transferrin preparations, the new high performance capillary technique was employed for isoelectric focusing. Iron‐free transferrin and transferrin samples of known iron content were examined. The above method gives an exceptionally rapid analysis (within 15–25 min) of small amounts of samples (less than 1 μg protein) and as good as or better resolution than other isoelectric focusing techniques previously used for transferrin analysis. By monitoring the focused protein zones at both 280 and 460 nm the molecular forms of transferrin (iron‐free, monoferric and differic complexes) can easily be identified. Both steps of isoelectric focusing in capillaries (i. e., prefocusing and mobilization) can be used for analysis. We observed that chelating agents (e. g., carrier ampholytes, nitrilotriacetate) may release iron from micro‐syringes having metal pistons causing the formation of iron‐transferrin complexes.
Improved separation of alpha chains of collagen type I, type III, and type V by noninterrupted electrophoresis using thioglycolic acid as a negatively charged reducer
Authors: Nakamura Kazuyuki , Inoue Shin‐Ichi , Abiko Seishou , Aoki Hideo , Takeo Kazusuke 
Published: May 2005

AbstractImproved separation of alpha chains of collagen type I (α1[I]2 α2[I]), type III(α1[III]3), and type V(α1[V] α2[V]α3[V]) was achieved by noninterrupted sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with a negatively charged reducer, thioglycolic acid. The thioglycolic acid, added to the running buffer of the cathodic reservoir, in the middle of electrophoresis quickly migrated in the gel anode, reducing interchain disulfide linkages in collagen type III and dissociating it into its alpha chain monomer, α1[III], without an interruption of electrophoresis. The alpha chain, α1[III], migrated more slowly than the α1[I] and α2[I] chains of collagen type I, resulting in an excellent separation of α1[III] from α1[I]. The mobility of α1[III] could be controlled by varying the time of thioglycolic acid addition to the running buffer. This enabled us not only to separate α1[III] from α1 [I] and α1[V], but also to precisely quantitate these alpha chains, even at low protein loading of mixed samples.
Enzyme activity electrophoresis and rocket immunoelectrophoresis for the qualitative and quantitative analysis of Geotrichum candidum lipase activity
Authors: Jacobsen Tomas , Poulsen Otto M. , Hau Jann 
Published: May 2005

AbstractThe development and application of a rocket immunoelectrophoretic and an enzyme activity electrophoretic assay for the quantitative and qualitative analysis of Geotrichum candidum lipase activity is presented. The sensitivities of the four assays were (in arbitrary units): enzyme activity electrophoresis, 1–0.5; rocket immunoelectrophoresis, 0.5–0.2; radial diffusion, 1; titrimetry, 1. The electrophoretic methods made it possible to distinguish between high and low molecular weight forms of the G. candidum lipases. The enzyme activity electrophoretic methods can be combined with other electrophoretic techniques, as demonstrated here with isoelectric focusing, and produce useful information on physico‐chemical differences between different molecular forms of the lipase, e. g. forms with different pI.
Effect of electric field switching on the electrophoretic mobility of single‐stranded DNA molecules in polyacrylamide gels
Authors: Lai Eric , Davi Noelle A. , Hood Leroy E. 
Published: May 2005

AbstractWe have examined the effects of pulsed electric fields on the separation of single‐stranded DNA molecules in polyacrylamide sequencing gels. Using different electric field pulsing regimens, the mobilities of single‐stranded DNA molecules can be retarded or increased as compared to conventional electrophoresis. These results indicated that pulsed field techniques can be applied to gel electrophoresis of small single‐stranded DNA molecules.
Protein database development: Identification of glycosylated and phosphorylated proteins in unfractionated rat fibroblast lysates
Authors: Krauss Marc R. , Collins Paul J. 
Published: May 2005

AbstractIn this paper, techniques are demonstrated for unambiguously mapping phosphoproteins and glycoproteins into a protein database of unfractionated fibroblast proteins. These methods allow for precise registration of subsets of proteins with the image of total proteins. The methods are also applicable to database mapping of antigens identified by indirect immunodetection methods and receptor molecules identified with photoaffinity ligands.
A simple method for accurately locating and cutting strips from the isoelectric focusing gel during two‐dimensional electrophoresis using the phastsystem
Authors: Schiff Theodore A. , Freedberg Irwin M. 
Published: May 2005

AbstractA rapid and simple method has been developed for precisely locating and cutting 1–3 mm strips from the isoelectric focusing gel during two‐dimensional electrophoresis using the PhastSystem. The protocol utilizes an easily constructed styrofoam template with marks to guide accurate cutting of the strips from the sample lanes. This system greatly enhances the reproducibility and quality of the two‐dimensional patterns obtained.
Modified diffusion blotting for rapid and efficient protein transfer with PhastSystem
Authors: Braun Wolfgang , Abraham Rolf 
Published: May 2005

AbstractAn easy‐to‐perform and efficient blotting method was developed for the transfer of proteins separated in PhastGel media. The method was evaluated by comparing the blotting results of low molecular weight marker proteins at different times. 14C‐Labelled proteins were used for the assessment of the transfer efficiency. Highly efficient transfer was achieved within 1 h of blotting and the proteins were almost completely eluted from the gel in 2 h. The gel remained on its solid plastic backing, which is not the case when using an electrophoretic transfer method. As little as 1.25 ng of electrophoretically separated protein were enzymatically detected on the blot when applying blot‐biotinylation. The transfer was performed at low temperature (4°C), which – in contrast to thermoblotting – additionally offers the chance to blot thermolabile proteins. These superior features, together with the method's versatility and low cost, mark an interesting alternative to the previously recommended blotting procedures after PhastSystem electrophoresis.
Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera
Authors: Tessmer Uwe , Dernick Rudolf 
Published: May 2005

AbstractViral polypeptides were prepared by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by copper staining and electroelution from gel slices. Poliovirus capsid polypeptide VP 1 isolated by this procedure induced monospecific antibodies in rabbits, i. e., antisera reacting only with the homologous polypeptide. Our results demonstrate the applicability of the described copper staining method as a rapid visualization step for preparing viral proteins after SDS‐PAGE.
Migration properties of circular DNAs using orthogonal‐field‐alternation gel electrophoresis
Authors: Hightower Robin C. , Santi Daniel V. 
Published: May 2005

AbstractThe migration of a series of supercoiled plasmids ranging in size from 4 to 91 kilobases (kb) has been analyzed by orthogonal‐field‐alternation gel electrophoresis (OFAGE). These circular DNAs enter OFAGE gels and are resolved over the same region of the gel as linear DNAs from 260 to 2200 kb. Furthermore, a distinct triphasic migration pattern was observed for the supercoiled DNAs. The migration of plasmids between 6 and 20, and 60 and 91 kb is inversely proportional to size, whereas the mobilities of plasmids between 20 and 60 kb increase with size. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a broad range of pulse times, from 10 to 120 s. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of small plasmids shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.
Temperature‐Gradient gel electrophoresis of nucleic acids: Analysis of conformational transitions, sequence variations, and protein‐nucleic acid interactions
Authors: Riesner Detlev , Steger Gerhard , Zimmat Rolf , Owens Robert A. , Wagenhöfer Manfred , Hillen Wolfgang , Vollbach Silke , Henco Karsten 
Published: May 2005

AbstractTemperature‐gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein‐nucleic acid interactions. A linear and highly reproducible temperature‐gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature‐gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand‐separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double‐stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (–)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch‐formation during the renaturation of (+)‐ and (−)strands from different clones. Examples are given for different viroid clones and HIV‐clones from one and the same patient. In another example, clones with point mutations from site‐directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the represser and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.
Demonstration of cell‐ricin interaction by electrophoretic light scattering
Authors: Hachemi Bahman Hakim , Betrencourt Claudine , Volochine Boris , Frénoy Jean‐Pierre , Chraibi Zakaria , Alfsen Annette , Lavialle FrançOise 
Published: May 2005

AbstractElectrophoretic light scattering has been used to investigate the interaction of ricin, a vegetal toxin, with cells. This technique allowed measurements in the presence of free ligand and proved particularly useful for the study of a system with low affinity. The electrophoretic mobility of erythrocytes and oligodendrocytes was found equal to 2.08 × 10−8 and 2.35 × 10−8m2s−1 V−1, respectively. Upon ricin binding, these values decreased significantly. This change was related to the saturation of the binding sites. The specificity of the interaction was demonstrated by conducting the experiments in the presence of lactose. This specific inhibitor fully prevented the ricin‐cell interaction.