bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pulsed field separation of large supercoiled and open‐circular DNAs and its application to bacterial artificial chromosome cloning
Authors: Wang Min , Lai Eric 
Published: May 2005

AbstractWe have studied the separation of large (80–300 kbp) supercoiled (SC) DNA in conventional agarose gel electrophoresis, field inversion gel electrophoresis (FIGE) and pulsed field gel electrophoresis (PFGE). DNA migration was measured under a variety of electrophoretic conditions including different switch times, temperatures, agarose concentrations, and voltage gradients. The migration of SC DNA was found to be inversely proportional to its molecular weight in the three electrophoresis systems tested. In conventional agarose electrophoresis, voltage gradient was found to be the determining parameter in the separation of SC DNA. Unlike large linear DNAs, the migration of SC DNA was found to be independent of switch time in PFGE and FIGE. Broad DNA bands were observed in prolonged FIGE runs. In addition, we have also studied the migration of open‐circular (OC) DNA (80 and 100 kbp) in pulsed field gel electrophoresis. Eighty kbp OC DNA can migrate into agarose gels under certain pulsed field conditions whereas 100 kbp OC DNA was trapped at the wells. Based on electrophoretic conditions described in this report, we can determine the size of bacterial artificial chromosome (BAC) clones without restriction enzyme digestion and have enriched the percentage of larger size clones in BAC cloning.
Single‐strand conformation polymorphism analysis by perpendicular temperature‐gradient gel electrophoresis
Authors: Sugano Kokichi , Fukayama Noriko , Ohkura Hisanao , Shimosato Yukio , Yamada Yasushi , Inoue Tamotsu , Sekiya Takao , Hayashi Kenshi 
Published: May 2005

AbstractUsing a newly developed temperature‐gradient gel electrophoresis apparatus, mutations in the K‐ras oncogene and p53 tumor supressor gene were analyzed for single‐strand conformation polymorphism (SSCP). The mobilities of single‐stranded DNAs, carrying various mutations, change – depending on the gel temperature during electrophoresis. Therefore, temperature‐gradient single‐strand conformation polymorphism (TG‐SSCP) analysis can provide useful information concerning the optimum temperature for SSCP and may also be used to screen for various mutations or polymorphisms in a single electrophoretic run.
Protein concentration by precipitation with pyrogallol red prior to electrophoresis
Authors: Marshall Thomas , Abbott Nicola J. , Fox Peter , Williams Katherine M. 
Published: May 2005

AbstractThe pyrogallol red protein assay (Clinical Chemistry 1986, 32, 1551–1554) is based upon formation of a blue protein‐dye complex in the presence of molybdate under acidic conditions. However, centrifugation of the assay mixture results in loss of color yield and precipitation of the protein‐dye complex which can be recovered and resolubilized to achieve protein concentration prior to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The method has been evaluated relative to trichloroacetic acid (TCA) precipitation for recovery and electrophoresis of commercial protein and peptide molecular weight markers. Precipitation with pyrogallol red‐molybdate (PRM) gives better and more uniform recovery of both proteins and peptides as compared to TCA. The lower limit of PRM precipitation is similar to TCA and corresponds to 1 μg protein per mL assay mixture. This is equivalent to 100 μL of 10 μg/mL protein using the standard protein assay or 1 μg/mL protein using a modified assay incorporating a fivefold concentrate of the dye reagent. Application of the method is demonstrated by concentration of urinary proteins. The method is simple and economic and useful for conserving trace amounts of precious sample as it allows recovery of protein for electrophoresis following protein assay.
Separation of arginase isoforms by capillary zone electrophoresis and isoelectric focusing in density gradient column
Authors: Pedrosa Mercedes M. , Legaz Maria Estrella 
Published: May 2005

AbstractFour major arginase isoforms, I, II, III and IV, have been detected in Evernia prunastri thallus. They differ in terms of both physical and biochemical properties. The isoelectric point (pI) of these proteins has been determined by both isoelectric focusing in density gradient column and high‐performance capillary electrophoresis (HPCE). Isoelectric focusing revealed charge microheterogeneity for isoforms II and IV whereas arginases I and II had the same pI value of 5.8. HPCE separation confirmed this charge microheterogeneity for isoform IV but not for isoform III, and provided evidence of microheterogneity for isoforms I and II. The effect of various electrolyte buffers and running conditions on the HPCE separation of arginase isoform were investigated. Addition of 0.5 mM spermidine (SPD) to the running buffer reduced the electroosmotic flow (EOF) and permitted discriminating between the native proteins and protein fragments.
Simple and sensitive laser‐induced fluorescence detection for capillary electrophoresis and its application to protein separation
Authors: Lim Heung Bin , Lee Jong Jin , Lee Kong‐Joo 
Published: May 2005

AbstractA low‐cost and highly sensitive laser‐induced fluorescence detector for on‐column detection systems was constructed and its applications were demonstrated in protein separation by capillary electrophoresis. The limit of detection (LOD) at a signal‐to‐noise ratio of 2 for Lissamine 20 was about 1.4 × 10−21 mole at 10 nL sample injection and the relative standard deviation (RSD) for 1.5 × 10−11 M of Lissamine 20 solution was about 15%. These values are comparable to or even better than those reported using a similar on‐column detection system. This sensitive LIF detection was applied for the study of tryptic digestion of insulin and for urinary protein profiling.
Indirect DNA/gene diagnoses via electrophoresis – an obsolete principle?
Authors: Epplen Jörg T. , Buitkamp Johannes , Epplen Cornelia , Mäueler Winfried , Rieß Olaf 
Published: May 2005

AbstractIn principle, gene defects can be investigated directly or indirectly via informative polymorphisms in their vicinity. But because many defects are not yet defined molecularly, these inherited diseases can only be diagnosed indirectly via analysis of informative family situations. Since (multiple) mutation analyses, e.g. via DNA sequencing, are time‐consuming and expensive, indirect analysis may still be performed initially – particularly in diseases caused by heterogenous mutations. We focus on diagnoses of neurological and (auto)immune diseases by polymerase chain reaction and separation of the DNA fragments via gel electrophoreses. Even after gene defects have been identified, indirect analysis might be necessary, for example in Huntington's chorea. Although this genetic defect has been characterized as a trinucleotide disease, indirect DNA diagnosis is still performed in particular cases for psychological reasons. The causes of autoimmune diseases are multifactorial and the inheritance is complex, involving several genes. Genome‐wide screening programs may involve indirect approaches via analyses of polymorphic microsatellites. Large parts of the immunological genome can be covered when 20 or more genes are investigated simultaneously. Thus the genetic bases of autoimmune diseases are disclosed. Microsatellites themselves could have a biological meaning. We therefore discuss also DNA/protein interactions for simple tandem repeats, the major targets for indirect gene diagnoses. Only indirect evidence exists that certain simple repeats influence genomic (in)stability. Taken together, indirect gene diagnoses supplement direct approaches in a variety of different purposes and in combination with standard electrophoresis techniques.
Commercial automated gel electrophoresis apparatus: Application to DNA, band dispersion, nonlinear Ferguson curves, and isolation
Authors: Chrambach Andreas , Yarmola Elena , Zakharov Sergey F. , Garner Mark M. 
Published: May 2005

AbstractRecently available commercial automated gel electrophoresis apparatus with intermittent scanning of fluorescently labeled gel patterns (the HPGE‐1000 apparatus of LabIntelligence, Menlo Park CA) was tested with regard to (i) its applicability to DNA in its native conformation, (ii) its ability to recognize the correct number of components, (iii) its capability to evaluate the width and shape of bands detected during electrophoresis, (iv) its ability to yield nonlinear Ferguson plots in a labor‐saving fashion, and (v) its preparative potential. Ethidium homodimer (EtD) DNA (bp) ratios were systematically varied and the mobility of DNA fragments labeled at each ratio was measured in order to find a ratio which provided an unaltered mobility and presumably therefore an unaltered conformation of the fragment. That ratio was found to be 1/40 EtD/ DNA (bp) or less. With such weak labeling of DNA, a representative fragment of 527 bp length requires a minimum load of 200 ng and a 2 μg load for a full‐scale peak height. Using the baseline automatically selected by the software of the apparatus, the band areas of the 17 components of a DNA digest were consistently evaluated by the software, as evidenced by the proportionality between DNA length and area. The areas of the separated bands of DNA fragments of 1857 and 121 bp length were found to be constant with time of electrophoresis. The dispersion coefficient was found to decrease with agarose concentration in electrophoresis at 1 V/cm; however, at higher field strength, the band width of the 1857 bp fragment was surprisingly found to increase with gel concentration, presumably due to stretching. Electrophoretic band dispersion was not found to be due to diffusion since zone spreading in the absence of the electric field under otherwise identical conditions does not proceed measurably. For the discontinuous buffer system of Wiltfang et al. (Electrophoresis 1993, 12, 352–366) the resolving power, in terms of theoretical plate equivalents, was approximately threefold higher compared to that in the Trisborate‐EDTA (TBE) buffer under otherwise identical conditions. Two electrophoretic runs of the apparatus with different gel concentrations in its eight channels suffice to define nonlinear Ferguson curves, provided that the free mobility is measured by capillary electrophoresis. The commercial automated apparatus excels as a preparative device by allowing one to monitor the qualitative and quantitative success of electroelution.
Biotinylation: A nonradioactive method for the identification of cell surface antigens in immunoprecipitates
Authors: Flesch Brigitte , Ntambi Elisabeth , Neppert Jürgen 
Published: May 2005

AbstractA nonradioactive method was employed to detect different cell membrane antigens on human polymorphonuclear granulocytes, monocytes and platelets. We compared the reactivity of one monoclonal antibody, N1III10, assumed to be FcγRII‐specific by functional assays, with other well‐characterized monoclonal antibodies and human sera. Intact cells were incubated with biotin N‐hydroxysulfosuccinimide ester which preferentially reacts with lysine residues in polypeptides. Biotin‐labeled cells were lysed and the antigen was isolated from the cell lysate by immunoprecipitation with the antibody bound to Protein A‐Sepharose. The precipitates were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and visualized by a streptavidin‐alkaline phosphatase system with a suitable substrate. Using this biotin‐labeling system we could show that N1III10 detects a 40 kDa antigen on monocytes and platelets, comparable to that expected of FcγRII monoclonal antibodies.
Separation and quantitation of reverse transcriptase polymerase chain reaction fragments of basic fibroblast growth factor by capillary electrophoresis in polymer networks
Authors: Gelfi Cecilia , Leoncini Fiorella , Righetti Pier Giorgio , Cremonesi Laura , di Blasio Anna Maria , Carniti Cristiana , Vignali Mario 
Published: May 2005

AbstractIn human ovarian carcinomas (epithelial and endometrial tumors) the presence of mRNA coding for the basic fibroblast growth factor (bFGF) was previously demonstrated by reverse transcription‐polymerase chain reaction (PCR). For quantitation purposes, competitive PCR is adopted, using as a competing fragment a sequence of a highly homologous bovine bFGF. However, separation and detection of the PCR products by slab gel electrophoresis and ethidium bromide staining gives poor quantitative data due to the nonstoichiometric binding of the dye. Thus, the only possible quantitation that can be obtained is via autoradiography with 32P‐labeled primers. We report here a capillary electrophoresis protocol, in 6% linear, liquid polyacrylamide as a sieving system, able to fully resolve and quantify the undigested (354 bp) bovine and the digested (295 bp and 59 bp) human fragments, with peak ratios in good agreement with the autoradiographic data.
A novel strategy to identify maternal and paternal inheritance in the mouse
Authors: Römer Irmgard , Jungblut Peter , Reik Wolf , Otto Albrecht , Klose Joachim 
Published: May 2005

AbstractA novel strategy for identifying proteins which reveal maternal or paternal inheritance in the mouse is presented. Using two‐dimensional electrophoresis we investigated protein expression patterns of adult liver and different embryonic and extraembryonic tissue in C57BL/6Crl and in DBA/2Crl mice, as well as in their reciprocal hybrids. We found three groups of protein spots which showed maternal or paternal inheritance of quantitative variations. These proteins were characterized by N‐terminal or internal amino acid sequencing, by determination of the amino acid composition, by glycoprotein staining and RNA expression analysis. The three proteins identified were: α‐enolase, cyclophilin and β‐group hemoglobins. The parental effects observed for α‐enolase and cyclophilin were found to be due to parent‐specific post‐translational modifications of these proteins. For the β‐group hemoglobins our results suggested parental effects on the transcriptional level.
Genotyping apolipoprotein B signal peptide insertion/deletion: A comparison of three methods
Authors: Glisic Sanja , Sunjevaric Ivana , Alavantic Dragan 
Published: May 2005

AbstractThree different ways of detecting insertion/deletion (Ins/Del) genotypes of the apolipoprotein B gene encoding signal peptide were compared. Unequal quantities of the two alleles were observed in 50 Serbian subjects. The low abundance allele was clearly visible only when a modified method of silver staining for polyacrylamide gels was used. This method is more reliable for Ins/Del polymorphism genotyping, avoiding misclassifications by conventional ethidium bromide staining of both agarose and polyacrylamide gels.
From image processing to classification: II. Classification of electrophoretic patterns using self‐organizing feature maps and feed‐forward neural networks
Authors: Keşmir Can , Søndergaard Ib , Jensen Kirsten 
Published: May 2005

AbstractIn a recent study, isoelectric focusing patterns were classified with a neural network using the back‐propagation algorithm [1]. In order to further study the classification process and to generalize the presentation of electrophoretic patterns, Kohonen's self‐organizing feature maps [2] were applied in this study. Although these feature maps are very efficient in many pattern recognition tasks, our data proved to be too complex for classification with an unsupervised system. Therefore, a second supervised network on top of the feature map was necessary. As in [3], a feed‐forward network trained by the back‐propagation algorithm was used. The final system allows us to correctly classify 90% of all wheat varieties. Moreover, the system proved to be reliable, reasonable in training time and shows the same accuracy in different experimental setups.
Imaging electrophoretic gels with a scanning beam laser macroscope
Authors: Seto Eileen K. , Damaskinos Savvas , Dixon Arthur E. , Diehl‐Jones William L. , Mandato Craig A. 
Published: May 2005

AbstractA scanning beam laser macroscopePatents pending.
has been developed which scans an area of 7.5 × 7.5 cm in 5 s. This new imaging system is examined as a potential tool for scanning electrophoretic gels. A specially‐designed telecentric, ƒ*θ laser scan lens is used in the instrument to achieve a linear scan and a flat focal plane. The laser scan lens focuses the incoming beam from a laser to a 10 μm spot inside the gel. A raster scan is performed across the gel and the signal is detected with a photomultiplier, forming a 512 × 512 digital image stored as a computer file. Silver‐stained protein polyacrylamide gels have been imaged in transmission and double‐transmission, while DNA agarose gels (stained with ethidium bromide) have been imaged in fluorescence with better than 25 pg. sensitivity. The macroscope has the advantage that it is not tied to the electrophoresis system as are end‐of‐line scanners, and the scan is rapid, so that several gels can be scanned in a very short time.
Free flow‐isoelectric focusing of human cellular lysates as sample preparation for protein analysis
Authors: Burggraf Dorothe , Weber Gerhard , Lottspeich Friedrich 
Published: May 2005

AbstractA whole cell lysate of human cells was separated into 80 fractions according to the pI of proteins using free flow isoelectric focusing with carrier ampholytes. The resolution of the process was highly reproducible, with an overlap of fractions of less than 30%. A protein of a faint silver stained spot in two‐dimensional gel electrophoresis (2‐DE) could be enriched, yielding a Coomassie blue stained spot which could be further characterized by proteinchemical methods. The enrichment of less abundant proteins from a complex crude cell extract was found to be a suitable tool for sample preparation and enrichment before applying proteins to 2‐DE and reversed‐phase high performance liquid chromatography.
Preparative reflux electrophoresis
Authors: Margolis Joel , Corthals Garry , Horvath Z. Stephen 
Published: May 2005

AbstractAs part of the method for large‐scale preparative electrophoresis across membranes of controlled porosity, we show that successive amounts of the leading component of a mixture migrating across a thin membrane can be collected by ‘reflux electrophoresis’. This consists of a series of cycles, in each of which the forward phase is stopped before the trailing components can emerge, the leading fraction is collected and the membrane cleared by reversing the current before commencing the next cycle. The reflux principle is demonstrated by separations based on size or on charge differences of protein molecules.
Separation of heat‐stable proteins from Thermus thermophilus HB8 by two‐dimensional electrophoresis
Authors: Kawaguchi Shin‐ichi , Kuramitsu Seiki 
Published: May 2005

AbstractThermostable proteins from Thermus thermophilus HB8, an extremely thermophilic bacterium, were separated by two‐dimensional gel electrophoresis. About 1200 spots were detected with silver staining on the gel between pH 3 and 10. According to the genome size of T. thermophilus, we consider that more than half of the proteins in the cell are visualized on a two‐dimensional gel. Using comigrated standard marker proteins, the molecular weight and isoelectric point of each protein spot were calculated. The average molecular weight and isoelectric point values were estimated to be 30 000 and 5.2, respectively. The average size and isoelectric point of detected protein from T. thermophilus were smaller and more acidic than those from Escherichia coli. After the protein spots had been electroblotted onto a polyvinylidene difluoride membrane and stained with Coomassie Brilliant Blue, the N‐terminal amino acid sequences were determined for about twenty protein spots. Few proteins had blocked N‐termini. Some spots were identified as proteins whose sequences had been reported previously from T. thermophilus. Others had amino acid sequences homologous with those of various proteins from other organisms. The amino acid sequence information of this report will be useful for obtaining stable proteins and for identifying open reading frames determined from the genome DNA sequence. Considering its small genome size and protein stability, T. thermophilus will be an excellent candidate for studying the molecular biology of an autotrophic living cell at the atomic level.
Two‐dimensional protein electrophoresis and multiple hypothesis testing to detect potential serum protein biomarkers in children with fetal alcohol syndrome
Authors: Robinson Mary K. , Myrick James E. , Henderson L. Omar , Coles Claire D. , Powell Mary K. , Orr Geoffrey A. , Lemkin Peter F. 
Published: May 2005

AbstractFetal alcohol syndrome (FAS) surveillance and intervention efforts are hampered by the lack of a specific biochemical test for diagnosis of the syndrome. Based on the hypothesis that abnormalities in growth and development (key features of FAS) involve altered protein metabolism, we analyzed serum proteins by two‐dimensional gel electrophoresis and image analysis to search for potential protein biomarkers of FAS. Serum samples from 12 participants in whom FAS had been diagnosed and 8 sex‐ and age‐matched participants whose mothers did not consume alcohol were analyzed in duplicate to determine whether the integrated intensities of matched proteins are significantly altered in children with FAS. Multiple hypothesis testing on 34 of the gels consisting of more than 1700 spots per gel revealed 21 proteins that we classified as potential protein biomarkers of FAS on the basis of significant t‐test differences at p < 0.02. We classified 8 of the proteins as candidate biomarkers on the basis of significant concentration differences between case and control subjects at p < 0.01. One of the proteins is clearly an isoform of retinol binding protein; two appear in the area of the gel where alcohol dehydrogenase is expected to appear; one appears to be an isoform of alpha‐1‐antitrypsin; three appear to be isoforms of the beta‐chain of haptoglobin; three may be forms of immunoglobulin light chains; and several others have not been associated with known proteins. No single protein differentiated all case subjects from control subjects, but stepwise canonical discriminant analyses revealed four groups of spots that distinguished between FAS case and control subjects with no misclassifications. Because of the small number of samples analyzed, we have not positively identified the proteins. It will be necessary to confirm our observations using additional samples before identifying each protein.