bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of rat plasma esterase‐1 (ES‐1) activity by scanning densitometry of gradient polyacrylamide gels with zymogram detection
Authors: Van Lith Hein A. , Haller Manuela , Van Zutphen Bert F. M. , Beynen Anton C. 
Published: May 2005

AbstractThere is no specific assay for rat plasma esterase‐1 (ES‐1) activity. Plasma contains many esterases, while known substrates do not discriminate between esterases. With gel electrophoresis, plasma esterase isozymes can be separated. Thus, a method consisting of gradient polyacrylamide gel electrophoresis, visualization of the enzyme with a staining technique based on substrate conversion, and densitometric scanning of the stained gel has been developed for quantitative measurement of rat plasma ES‐1 activity. ES‐1 activities were expressed as total peak areas. Reproducibility of the method was found to be about 10% (expressed as apparent between‐gel coefficient of variation). When the ES‐1 zone areas was expressed relative to that of a plasma ES‐1 standard, reproducibility was about 3%. The kinetics of catalysis of α‐naphthyl acetate hydrolysis by ES‐1 could be determined with the gel scanning assay; the Km was 0.76 mm. At the α‐naphthyl acetate concentration of 2.69 mm, total peak areas of the ES‐1 zone were linearly associated with the staining time (up to at least 40 min) and amount of plasma (up to 26.25 μL). The pH of the staining buffer influences the ES‐1 zone area, the largest areas being obtained when the pH ranged between 7.0 and 7.8. With propionate as acyl moiety of the α‐naphthyl ester substrate, ES‐1 zone areas were higher than with either acetate, butyrate or hexanoate.
A new staining method for the detection of activities of H2O2‐producing oxidases on gels and blots using cerium and 3,3′‐diaminobenzidine
Authors: Seitz Jürgen , Keppler Claudia , Fahimi H. Dariush , Völkl Alfred 
Published: May 2005

AbstractCerium chloride (CeCl3) was used to trap the hydrogen peroxide generated by several oxidases on native gels and blots. The pale yellow color of cerium perhydroxide formed is converted to a brown‐black precipitate by the subsequent reaction with 3.3′‐diaminobenzidine. The suitability of this method for the detection of the activity of several oxidases on gels and on blots under nondenaturing conditions, employing different electrophoretic systems and resolving techniques, is demonstrated. Moreover, this method has proven to be highly suitable for the assessment of the substrate and stereospecifity of oxidases, the determination of their molecular weights, and the isoelectric points of isoforms.
Pressure refilled polyacrylamide columns for the separation of oligonucleotides by capillary electrophoresis
Authors: Sudor Jan , Foret František , Boček Petr 
Published: May 2005

AbstractThe separation of oligonucleotides by capillary zone electrophoresis (CZE) was studied in fused silica separation capillaries filled by linear (noncrosslinked) polyacrylamide (PAA) solutions, introduced into the capillary from the stock by pressure after each analysis. The time‐consuming in‐capillary polymerization step could thus be avoided, and fast and reproducible repetition of the analyses was assured. The PAA concentrations varied within the range of 3–10% and both the reproducibility of the analyses and the stability of the solution in the capillary, with and without a chemically treated inner wall, were tested. Ferguson plots were used to assess the size selectivity of the separation.
High speed electrophoresis simulation for optimization of continuous flow electrophoresis and high performance capillary techniques: Part I. Computer model
Authors: Heinrich Jörn , Wagner Horst 
Published: May 2005

AbstractA computer program for high‐speed simulation and optimization of electrophoretic processes has been developed for carrier‐free systems of all kinds. The calculations are based on the one‐dimensional dynamic (transient‐state) model. The three‐dimensional geometry of the simulation space can be chosen deliberately. With a highly efficient transport algorithm instead of complicated integration schemes for the transport equations, the calculation time can be effectively spent on various important parameters such as ionic strength, temperature, Joule heat, activity coefficients and concentration changes due to membranes. The parameter set of any carrier free electrophoretic method (i.e., continuous‐flow electrophoresis, capillary isotachophoresis and high performance capillary zone electrophoresis) can be imported directly into the computer program by means of a graphic user interface. The program performs overnight‐simulation of any electrophoretic system containing up to 15 components.
Formamide modified polyacrylamide gels for DNA sequencing by capillary gel electrophoresis
Authors: Rocheleau Marie J. , Grey Ronda J. , Chen Da Yong , Harke Heather R. , Dovichi Norman J. 
Published: May 2005

AbstractCompressions are occasionally found during the separation of DNA sequencing fragments, particularly in G/C‐rich regions and in gels operated at room temperature. Addition of at least 10% formamide to urea/polyacrylamide sequencing gels improves the denaturing capacity of the gel, minimizing compressions. Addition of 20% or more formamide decreases the separation rate, theoretical plate count, and resolution for normally migrating fragments. An optimum concentration of 10% formamide improves resolution of compressed regions without degrading the other characteristics of the gel. Operation of gels at room temperature simplifies the engineering associated with automated sequencers based on capillary gel electrophoresis.
Pulsed field sequencing gel electrophoresis
Authors: Brassard Eric , Turmel Chantal , Noolandi Jaan 
Published: May 2005

AbstractThe effect of pulsed fields on sequencing gel electrophoresis is investigated, using DNA fragment markers ranging in size from 20 to 6557 bases. For high continuous electric fields (5000 V/55 cm) band inversion is observed in which fragments larger than 4000 bases migrate faster than those of 800–1000 bases. The use of one‐dimensional pulsed field gel electrophoresis (ODPFGE) eliminates band inversion and extends the monotonic size‐mobility relationship of the DNA markers up to about 4000 bases. The relevance of these results, obtained using a manual sequencing process with autoradiographic detection, to automated sequencers is discussed.
Analysis of members of the Ig‐gene superfamily by thermal gradient polyacrylamide gel electrophoresis
Authors: Tulp Abraham , Verwoerd Desireé 
Published: May 2005

AbstractA solid aluminum block, connected with a warm and cold thermostated waterbath, provided for a linear transversal temperature gradient (TG) during polyacrylamide gel electrophoresis (PAGE). Noncovalently bound heavy chain dimers as well as heavy‐light chain dimers, derived from human monoclonal IgG, could be melted into monomers using a 40–75°C TG under conditions of sodium dodecylsulfate‐PAGE. Using native PAGE, majorhistocompatibility complex (MHC) class I molecules, preloaded with the iodinated peptide FAPGNYPAL could be melted in a 4–40°C TG to release the peptide. The method is in general applicable to thermal stability analysis of noncovalently bound hetero‐oligomers if the product after melting possess different electrophoretic mobilities.
Thin‐layer electrophoresis with PhastSystem facilitates analysis of phosphoamino acids from proteins bounc to Immobilon
Authors: Lippmann Corinna , Lindschau Carsten , Erdmann Volker A. 
Published: May 2005

AbstractA modification of thin‐layer electrophoresis of phosphoamino acids on miniplates, using the PhastSystem (Pharmacia) is described. This method not only reduces the amount of radioactivity required but also the radioactive waste. In addition, it also decreases the handling of hazardous substances and takes only a fraction of the time required for the standard procedure.
Preparative continuous separation of biological particles by means of free‐flow magnetophoresis in a free‐flow electrophoresis chamber
Authors: Hartig Roland , Hausmann Michael , Schmitt Joachim , Herrmann Dieter B. J. , Riedmiller Michael , Cremer Christoph 
Published: May 2005

AbstractFor sorting, cells or cellular components can specifically be labeled by antibody‐coated magnetic beads. We have developed a device for continuous magnetic sorting based on the flow‐chamber of a free‐flow electrophoresis system. Magnetically labeled particles are injected into a given continuously flowing chamber buffer and pass an inhomogeneous magnetic field, configurated perpendicular to the flow direction. According to its magnetic moment, the magnetic material is deviated into the direction of the magnetic forces, while nonmagnetic material passes the field without interaction. The magnetic forces can be changed with the electrical current of the solenoids producing the magnetic field. As in the free‐flow electrophoresis system, the particle fractions are collected in different vials. Online control of the experiments can be performed by an optical scanning system. Experiments with model particles achieved a sorting purity of more than 99% at a rate of up to 5 × 108particles per hour. In experiments with blood cells, a high enrichment of either B‐or T‐lymphocytes was obtained. In contrast to free‐flow electrophoresis, there is no limitation, in principle, regarding the type of chamber buffer to be used. This allows an optimal adaptation of the buffer conditions to the requirements of vital sorting. The preliminary results so far confirm this conclusion.
Application of capillary electrophoresis in peptide research
Authors: Yildiz Eraslan , Grübler Gerald , Hörger Susanne , Zimmermann Hans , Echner Hartmut , Stoeva Stanka , Voelter Wolfgang 
Published: May 2005

AbstractComparison of high performance liquid chromatograms (HPLC) with capillary electrophoresis shows the latter to be superior in many cases owing to rapid separation, high resolution and high sensitivity. This is demonstrated with two examples (i) the isolation of natural peptides from bovine tissue and (ii) characterization of a synthetic peptide mixture with the natural sequence (fragment) of the human immunodeficiency virus (HIV) transmembrane glycoprotein gp 41 env.
Improving the detection of proteins after transfer to polyvinylidene difluoride membranes
Authors: Sanchez Jean‐Charles , Ravier Florence , Pasquali Christian , Frutiger Séverine , Paquet Nicole , Bjellqvist Bengt , Hochstrasser Denis F. , Hughes Graham J. 
Published: May 2005

AbstractN‐Terminal sequence analysis of proteins separated by two‐dimensional polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes has become the method for molecular characterization of proteins contained in biological samples. However, the proteins of lower abundance cannot be sequenced directly, without improving the technique. We have studied a drying method on several PVDF membranes including Trans‐Blott, Immobilon P and Problott. Using Amido Black, Coomassie Brilliant Blue R‐250 and Ponceau S, we have obtained, in comparison with the non‐dried membranes, an enormous increase in the number of detectable proteins.
Examination of microheterogeneity in grass pollen allergens
Authors: Petersen Arnd , Becker Wolf‐Meinhard , Schlaak Max 
Published: May 2005

AbstractThe separation of timothy pollen extract by two‐dimensional immunoblotting revealed microheterogeneity of the major allergens PhI p I and PhI p V. There was not only a diversity in size, 38 and 32 kDa for PhI p V and 37,35, and 32 kDa for PhI p I, but also a separation into proteins of identical sizes but different pIs. Since former studies on the protein structure by amino acid analysis and N‐terminal micro‐sequencing did not reveal any differences, we examined other possibilities that might cause microheterogeneity. In allergens belonging to the PhI p I group, the variability in pI can be due to the carbohydrate structure and to the fact that charges are hidden in the interior of the protein, as shown by varying concentrations of urea. On the other hand we were not able to detect any such reasons for the existence of PhI p V isoallergens. Thus, we assume that they are stable conformational isomers of the proteins (allomorphism) or that there are only slight variations in the internal sequences of these proteins (polymorphism) causing distinct pIs.
Detection of polypeptides and amylase isoenzyme modifications related to malting quality during malting process of barley by two‐dimensional electrophoresis and isoelectric focusing with immobilized pH gradients
Authors: Görg Angelika , Postel Wilhelm , Weiss Walter 
Published: May 2005

AbstractTwo cultivars (“Alexis” and “Lenka”) of contrasting final attenuation values were malted, and the protein and amylase isoenzyme composition, as well as the change in protein and amylase isoenzyme composition during malting, was investigated by two‐dimensional polyacrylamide gel electrophoresis of total proteins, and isoelectric focusing of amylase isoenzymes, respectively. Isoelectric focusing demonstrated that significant differences exist between the amylase isoenzyme patterns of the two cultivars, suggesting a correlation between the presence of certain amylase isoenzyme bands and final attenuation. This finding was confirmed by analysis of 36 barley cultivars with a wide range of quality. It was shown that all cultivars which are of low or, at best, moderate final attenuation values exhibit the amylase band “B” (isoelectric point ≈︁ 6.8), whereas those cultivars which are predominantly of high malting grade do not possess this “B” isoenzyme band, but exhibit the pronounced “A” isoenzyme band (isoelectric point ≈︁ 6.5) instead, suggesting that these isoenzymes (which we suppose to be β‐amylases) can be utilized to predict the final attenuation values of unknown barley samples or new lines. However, “final attenuation” is a complex function. Preliminary results of two‐dimensional gel electrophoresis indicate that other factors, such as total amount of amylases, or a 19 kDa A hordein‐like polypeptide, which was degraded faster in the low malting grade cultivar “Lenka”, may also have a role in determining quality.
Improved phenotyping of alpha1‐antichymotrypsin (ACT) by isoelectric focusing and immunoprinting: First demonstration of a deficient protein variant in the ACT system
Authors: Weidinger Sebastian , Poller Wolfgang , Faber Jakob‐Peter , Schwarzfischer Friedrich 
Published: May 2005

AbstractGenetic variation of human alpha1 ‐antichymotrypsin (ACT) was investigated in sera using thin‐layer polyacrylamide gel isoelectric focusing (pH range 4.0–6.5) followed by immunoprinting with a monospecific anti‐human ACT antibody. Sialidase‐treated samples showed a microheterogeneous banding pattern which consisted of two major and several additional minor components with isoelectric points between pH 5.0 and 5.3. A population study of 200 unrelated individuals from southern Germany revealed no genetic variation. In a clinical investigation, however, we found a unique banding pattern in a female patient suffering from chronic obstructive pulmonary disease. In comparison with the monomorphic normal type the detected variant phenotype shows two additional bands that have lower intensities and are located cathodically to their major bands. Inheritance of the deficient IEF variant “ACT Bochum” was confirmed by a family study. To our knowledge this is the first genetic ACT mutant to be observed at the protein level.
Isoelectric focusing and immunoblotting of the platelet membrane glycoprotein complex IIb. IIIA following urea solubilization
Authors: Gianazza Elisabetha , Frattini Roberta , Michelagnoli Silvia , Cassinotti Michela , Sirtori Cesare R. 
Published: May 2005

AbstractEffective solubilization of the major platelet membrane component, the glycoprotein IIb. IIIa complex, can be achieved with 8 M urea. By avoiding nonionic detergents in the separation medium it is possible to obtain clear immunoblot patterns without interference from the isoelectric focusing matrix. Upon running on a pH 4.25–5.25 immobilized pH gradient, immunoreactive bands corresponding to the nonreduced IIb.IIIa complex stain between pH 4.5 and 5.0. The method appears of significant potential utility in evaluating glycoprotein IIb.IIIa polymorphisms under different clinical conditions.
Genetic study of orosomucoid by isoelectric focusing and immunoprinting in patients with carcinoma
Authors: Mittermüller Johann , Weidinger Sebastian 
Published: May 2005

AbstractThe carbohydrate moiety of the orosomucoid (ORM) molecule shows microheterogeneity [1] and the pteridine‐containing variant seems to be tumor‐specific [2–4]. However, there also exists a genetic (protein‐related) polymorphism coded by the ORM1 and ORM2 loci on chromosome 9 [5, 6]. To investigate the relationship between ORM1 gene products and the development of carcinoma, we analyzed the ORM1 phenotypes of desialated sera 125 patients with carcinoma. The allele frequencies were estimated for ORM1*F1 0.556, ORM1*F2 0.012 and ORM1*S 0.432. In comparison to healthy individuals from the same geographical area [6] the ORM1 S phenotypes are significantly more frequent in carcinoma patients. The patients' sera frequently showed additional ORM‐positive proteins which focused slightly cathodically to the ORM 2 A band. These proteins may represent posttranslational modifications of the ORM1*S allele product. Whether these modifications are tumor‐specific and related to the carbohydrate moiety of the molecule must be confirmed in further studies.
Superimposing two‐dimensional gels to study genetic variation in malaria parasites
Authors: Horgan Graham , Creasey Alison , Fenton Brian 
Published: May 2005

AbstractTwo‐dimensional polyacrylamide gel electrophoresis is a valuable tool for studying genetic variation in the human malaria parasite, Plasmodium falciparum. It involves examining the positions of protein spots in gels produced from different isolates. Some spots have been seen to vary, while others have had a constant position in all isolates so far examined. These invariant spots provide a reference frame to compare variations in other spots. This paper discusses the usefulness of digital image handling, warping and superimposition in a personal computer environment. Rather than produce a fully automatic interpretation system, we show how the computer may be used as a tool for manipulating gel images, although interpretation of the gels' features remains with the human expert. Autoradiographs are scanned on a desktop scanner, and the images in digital form can be displayed on a monitor attached to a personal computer. The coordinates of the invariant spots on each of several gels are identified by the user. Each of the gels is then warped so that the invariant spots of all the gels coincide as closely as possible. The variable spots are then examined. We have used both affine warping transformations, which match the invariant spots as closely as possible, and thin plate spline transformations, which match them exactly. Colour superimposition proved a useful way of examining the gels.
A simple and rapid method for HLA‐DQA1 genotyping by polymerase chain reaction‐single strand conformation polymorphism and restriction enzyme cleavage analysis
Authors: Hayashi Tomonori , Seyama Toshio , Ito Takashi , Kusunoki Yoichiro , Hirai Yuko , Nakamura Nori , Akiyama Mitoshi 
Published: May 2005

AbstractA simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)‐DQA1 locus is described. The polymorphic second exon of the HLA‐DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single‐strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, he eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA‐DQA1 genotypes should also be possible.
Microsequences of 145 proteins recorded in the two‐dimensional gel protein database of normal human epidermal keratinocytes
Authors: Rasmussen Hanne H. , Van Damme Josef , Puype Magda , Gesser Borbala , Celis Julio E. , Vandekerckhove Joël 
Published: May 2005

AbstractMicrosequencing of proteins recovered from two‐dimensional (2‐D) gels is being used systematically to identify proteins in the master human keratinocyte 2‐D gel database. To date, about 250 protein spots recorded in human 2‐D gel databases have been microsequenced and, of these, 145 are recorded in the keratinocyte database under the entry partial amino acid sequence. Coomassie Brilliant Blue‐stained protein spots cut from several (up to 40) dry gels were concentrated by elution‐concentration gel electrophoresis, electroblotted onto PVDF membranes and digested in situ with trypsin. Eluting peptides were separated by reversed‐phase HPLC, collected individually and sequenced. Computer search using the FASTA and TFASTA programs from Genetics Computer Group indicated that 110 of the microsequenced polypeptides shared significant similarity with proteins contained in the PIR, Mipsx or GenEMBL databases. Only 35 polypeptides corresponded to hitherto unknown proteins. Peptide sequences of all 145 proteins are listed together with their coordinates (apparent molecular weight and pI) in the keratinocyte database.
Human cerebrospinal fluid protein database: Edition 1992
Authors: Yun Miki , Wu Wei , Hood Lee , Harrington Michael 
Published: May 2005

AbstractTwo‐dimensional electrophoresis maps of human cerebrospinal fluid proteins are presented in the form of labeled images. 931 protein spots are identified in spinal fluid from a normal volunteer. Distinct spots that represent variants of the same protein, especially posttranslational modifications, are estimated to reduce the 931 different spots to < 200 different proteins. 248 spots of 29 protein groups have been identified and are indicated on enlargements of specific gel regions. The distribution of protein abundance, mass, charge and shape characteristics of these normal 931 spinal fluid spots are graphically profiled. Analysis of the shape parameter “vertical height: width ratio” reveals that a ratio > 3.5 correlates with glycoproteins, enabling their identification simply by image analysis. Proteins that are not present on the normal map, but appear in spinal fluid in patients with schizophrenia and Creutzfeldt‐Jakob disease are illustrated on additional maps.
Workshop on two‐dimensional gel protein databases
Authors: Simpson Richard J. , Tsugita Akira , Celis Julio E. , Garrels James I. , Werner Mewes H. 
Published: May 2005

AbstractA workshop on two‐dimensional gel electrophoresis (2‐DE) protein database, organized by the Committee on Data for Science and Technology (CODATA) of the International Council of Scientific Unions Task Group on Biological Macromolecules, was held at the CODATA Secretariat in Paris on March 9, 1992. Eleven scientists from eight different countries represented various aspects of 2‐DE analysis – namely, cellular protein database development and protein microsequencing methodologies. The purpose of the workshop was to explore means of integrating the rapidly expanding body of information on 2‐DE resolved proteins from different laboratories. A major proposal emanating from the workshop was the establishment of an intermediary or “relational” 2‐DE gel protein database. This intermediary database, which would catalogue pertinent information on 2‐DE resolved proteins (experimental source, 2‐DE loci, biological information, etc.) could be an adjunct to, and accessed through, the existing international protein sequence databanks. It would function as a pointer for researchers to the individual 2‐DE protein databases where primary and more specialized 2‐DE data would be housed.