bluuebunny/crossref_metadata_2025_split huggingface dataset:
Separation of DNA sequencing fragments using an automated capillary electrophoresis instrument
Authors: Karger Achim E. 
Published: May 2005

AbstractRapid, high‐resolution separation of DNA sequencing fragments by capillary gel electrophoresis using an automated, commercially available instrument is presented. The effect of column lengths and electric field strength on the resolution of sequencing fragments as well as the sensitivity of laser‐induced rescence (LIF) detection was investigated. Using a short capillary of 20 length, which results in a U‐shape of the capillary in the capillary cartridge very high separation efficiency, up to 17 × 106 theoretical plates per m, is obtained. Analysis of the band broadening factors revealed that the resolution on the short column is predominantly determined by axial diffusion and to a minor extent by detection zone width. Presumably due to the coiling of longer capillaries in the capillary cartridge, increasing the capillary length does increase the separation efficiency as predicted for diffusion‐limited separation. The concentration limit to detection (signal‐to‐noise ratio = 2) is 0.2 × 10−1 M of fluorescein‐labeled oligonucleotide primer under the separating conditions for DNA sequencing samples. Increasing the electric field strength from 100 to 175 V/cm improved resolution and at the same time approximately doubled the sequencing speed. Fragments up to 500 nucleotides in length are resolved in less than 50 min.
Capillary zone electrophoresis and micellar electrokinetic chromatography, with taurodeoxycholate as micellar agent, of protein kinase A peptide substrates
Authors: Beijersten Ingegerd , Westerlund Douglas 
Published: May 2005

AbstractThe separation of protein kinase A peptide substrates with the general formula ‐X‐Arg‐Arg‐Ala‐Ser‐Y‐, where X and Y may be the same or different amino aicds, was studied by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Taurodeoxycholate (TDC) was used as the micellar agent. CZE was effective in separating a peptide series differing in the number of amino acids, but not for a series with a difference in the terminating amino acid. For the latter series, MEKC generally gave a higher selectivity, but some of the peptide pairs were more easily separated by CZE, demonstrating the complementary character of the two techniques. The efficiency of the MEKC system was typically < 50% of that of CZE, but its higher selectivity generally outbalanced the lower efficiency regarding resolution. The distribution of the peptides to the micelles was studied by determination of retention factors. Electrostatic and hydrophobic forces were found to be determining factors in the distribution; the most highly charged basic peptides were most heavily distributed, and for peptides with the same charge those containing more hydrophobic amino acids were more strongly distributed. The contribution of some structural features to the distribution degree was also determined.
High resolution density gradient electrophoresis of cellular organelles
Authors: Tulp Abraham , Verwoerd Desirée , Fernandez‐Borja Mar , Neefjes Jacques , Hart Augustinus A. M. 
Published: May 2005

AbstractA density gradient electrophoresis apparatus made of Perspex was constructed, with a separation column (7 × 2.2 cm) containing a 0–5% linear Ficoll gradient. The useful separation path is 6 cm. A specially designed gradient mixer is described which fits over the application cone. This cone permits precise gradient and sample introduction as well as undisturbed fractionation after electrophoresis. A bottom circular palladium cathode is separated hydrodynamically but not electrically from the density gradient by a cellophane membrane, merely secured by an O‐ring. The top circular platium anode allows for upward electrophoresis (80–100 min at 10 mA). The markedly higher resolution of subcellular organelles was compared with separations obtained earlier with a small, but much more difficult to fabricate, prototype. Moreover, ease of manipulation was greatly improved. A wide separation distance was obtained between plasma membrane, endoplasmatic reticulum as well as between two populations of lysosomes. Even early, middle, and late endosomes could be separated with high resolution. Soluble isoenzymes could be separated as well and were far away from the vesicle‐enclosed enzymes.
Chemiluminescent enhanced CD47 detection on Western blotting
Authors: Yuan Fang Fang , Mihrshahi Seema , Fletcher Anne 
Published: May 2005

AbstractCD47 is a leucocyte antigen of wide tissue distribution including platelets and erythrocytes. It has been found that anti‐CD47 antibodies may cause platelet activation. Sensitive techniques are therefore needed for biochemical investigation of platelet glycoproteins including CD47. Conventional Western blotting techniques using horseradish peroxidase‐conjugated antibody and 3,3′‐diaminobenzidine (DAB) substrate resulted in unsatisfactory results with CD47 due to low sensitivity. When chemiluminescent detection with horseradish peroxidase‐conjugated antibody was used in conjunction with Western blotting, detection of the CD47 molecule was greatly enhanced. A protein of 47–55 kDa was immunoblotted from platelets by anti‐CD47 while the mAb immunoblotted a broader band (45–60 kDa) from erythrocyte membranes. Thus, there may be different forms of CD47 present on platelets and erythrocytes. The results indicate that Western blotting with the chemiluminescent detection technique is a useful and sensitive alternative for the study of human platelet antigens.
Analysis of recombinant and native CD4 by one‐ and two‐dimensional gel electrophoresis
Authors: Lynch Garry W. , Dearden Mark , Sloane Andrew J. , Humphery‐Smith Ian , Cunningham Anthony L. 
Published: May 2005

AbstractKnowledge of CD4 conformation within the membranes of human lymphoid and monocytoid cells is essential for a clear understanding of its function as a ligand for major histocompatibility complex II (MHC) molecules in T cell activation and for gp120 in human immunodeficiency virus (HIV) infection. The charge and structure of native (nCD4) and soluble recombinant CD4 (rCD4) were examined by one‐ and two‐dimensional (2‐DE) electrophoresis antigen mapping and silver staining. Recombinant CD4 was partitioned by nonequilibrium pH gradient electrophoresis (NEPHGE) and revealed a number of differentially charged 44 kDa species (pI > 9.5). Biotinylation (4 h, room temperature) of rCD4 yielded a single labelled species on sodium dodedyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) with an increased apparent molecular mass to 50 kDa, consistent with a maximal incorporation of ∼ 18 molecules of biotin per rCD4 molecule. The milder biotinylation (15 min, 4°C) of cell‐(CEM‐T4, THP‐1) expressed CD4 was not accompanied by any apparent alteration in molecular weight, nor abrogation of CD4 antigenicity. This was determined by isolation of nCD4 by immunoprecipitation and SDS‐PAGE immunoblotting, using anti‐CD4 mAbs (leu3a, OKT4A, Q4120, T4, OKT4, Q425) and by flow cytometry (leu4a, T4). The immunoprecipitation of full‐length native CD4 from lymphoid MT2 and CEM‐T4 cell extracts, however, revealed both monomeric and higher‐order CD4 antigen complexes by immunoblotting. These studies describe the biotinylation, 1‐DE and 2‐DE of CD4 preparations, and indicate the capacity of CD4 of lymphocytes to form complexes which may influence CD4 conformation and epitope availability.
Different electrophoretic techniques produce conflicting data in the analysis of myocardial samples from dilated cardiomyopathy patients: Protein levels do not necessarily reflect mRNA levels
Authors: Dos Remedios Cristobal G. , Berry Desirée A. , Carter Laura K. , Coumans Joëlle V. F. , Heinke Monique Y. , Kiessling Peter C. , Seeto Reginald K. , Thorvaldson Tom , Trahair Toby , Yeoh Thomas , Yao Mu , Gunning Peter W. , Hardeman Edna , Humphery‐Smith Ian , Naidoo Daya , Keogh Anne 
Published: May 2005

AbstractA variety of electrophoretic techniques were used to search for potential causes of human dilated cardiomyopathy (DCM). Northern blots were used to quantify α‐cardiac and α‐skeletal muscle actins, and β‐myosin heavy chain mRNAs which are the predominant expressed isoform species. We found a wide range of mRNA levels expressed in both DCM and nondiseased (ND) samples of left ventricles. However, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels of the same heart samples revealed a stable and constant ratio of actin and myosin. Dystrophin deficiency might account for the DCM symptoms and so dystrophin levels of DCM and ND samples were evaluated using Western blots probed with monoclonal antibodies for the N‐, C‐ and mid‐rod portions of this protein. We found that dystrophin levels were constant in all 29 DCM and 5 ND samples suggesting that dystrophin deficiency is probably not a contributing cause. We explored the possibility that terminal failure may be due to an apoptotic‐like event in the cardiomyocytes. Zymograms of DCM and ND samples revealed a significant increase in DNase I activity in the DCM group compared to the ND samples. These data raise the possibility that end‐stage failure may be associated with apoptosis.
Genetic screening and testing by induced heteroduplex formation
Authors: Wood Nigel , Bidwell Jeffrey 
Published: May 2005

AbstractClustered point mutations and small deletions or insertions within DNA are amenable to rapid analysis using induced heteroduplex formation. A single synthetic molecule (universal heteroduplex generator, UHG) may detect any of a series of such mutations following amplification by the polymerase chain reaction. This paper illustrates the use of UHG‐based DNA heteroduplex analysis by describing the construction, properties, and methods of use of five UHGs, designed for genetic screening and testing of the inherited metabolic diseases: phenylketonuria, sickle‐cell disease, cystic fibrosis, von Willebrand's disease type 2B, and mamman‐binding lectin deficiency. In all cases, identification of multiple disease‐associated genotypes is possible using a single UHG.
Capillary electrophoresis with pulsed amperometric detection of carbohydrates and glycopeptides
Authors: Weber Paul L. , Lunte Susan M. 
Published: May 2005

AbstractThe utility of capillary electrophoresis with pulsed amperometric detection (CE‐PAD) for the analysis of carbohydrate‐containing samples from a variety of biological sources is described. CE‐PAD was used to separate a mixture of oligosaccharides obtained from bovine fetuin and to monitor the desialylation process used in the characterization of the oligosaccharides. Additionally, the high resolving power of the system was demonstrated using a series of glycopeptides obtained from recombinant coagulation factor VIIa, which possess the same decapeptide core but differ in the extent of sialylation. Deglycosylation of these glycopeptides for characterization purposes resulted in a mixture of carbohydrates and peptides. Unlike CE with UV detection, this system gave good responses for all analytes, demonstrating the unique ability of PAD to respond to the electrochemical features of diverse classes of biomolecules such as carbohydrates and peptides. Finally, CE‐PAD was applied to the analysis of a tryptic digest of recombinant tissue plasminogen activator. The use of different detection potentials in sequential runs on a sample gave structural information about the peptides, such as glycosylation. A brief review of prior applications of CE‐PAD to the analysis of standard mixtures of simple saccharides is also presented.
Separation of 8‐aminonaphthalene‐1,3,6‐trisulfonate (ANTS)‐labeled oligomers containing galacturonic acid by capillary electrophoresis: Application to determining the substrate specificity of endopolygalacturonases
Authors: Mort Andrew J. , Chen Emily Min Wu 
Published: May 2005

AbstractSlight modifications of published procedures (Stefansson, M. and Novotny, M., Carbohydr. Res. 1994, 258 1–9) allow separation by relative size of 8‐aminonaphthalene‐1,3,6‐trisulfonate (ANTS)‐labeled neutral and acidic oligosaccharides by electrophoresis in uncoated capillaries. The separations are performed at pH 2.5 to suppress both any charge from carboxylic acid groups on the oligosaccharides and electroosmotic flow. Utility of the procedure is demonstrated by its application to characterization of substrate specificity of endopolygalacturonases. The results show that both a fungal and a bacterial endopolygalacturonase need four adjacent nonesterified galacturonic acid residues in a pectin to be able to act.
High performance capillary electrophoresis method to characterize heparin and heparan sulfate disaccharides
Authors: Karamanos Nikos K. , Vanky Peter , Tzanakakis George N. , Hjerpe Anders 
Published: May 2005

AbstractA rapid, sensitive and accurate high‐performance capillary electrophoresis method is described for the determination of the sulfation pattern of heparin and heparan sulfate disaccharides. The analysis, performed after enzymic degradation of the polysaccharides with heparinase and heparinases II and III in combination, yields highly UV‐absorbing Δ‐disaccharides. The separation is performed with reversed polarity using 15 mM phosphate buffer, pH 3.50. This method is superior to others since all known 12 disaccharides carrying N‐acetylated, N‐sulfated or unsubstituent glucosamine can be separated in a single run of 15 min. At the highest sensitivity the analysis consumes only a few femtograms of glycosaminoglycan and allows a determination of Δ‐disaccharides at the attomole level.
Profiling of oligosaccharide‐mediated microheterogeneity of a monoclonal antibody by capillary electrophoresis
Authors: Hoffstetter‐Kuhn Sabrina , Alt Gerlinde , Kuhn Reinhard 
Published: May 2005

AbstractBased on complex formation of borate with carbohydrates in alkaline solutions, the oligosaccharide microheterogeneity of a monoclonal antibody was studied using capillary zone electrophoresis. In borate buffers characteristic separation patterns were found that could be attributed to the same antibody by their UV spectra, while in a phosphate buffer, under otherwise the same conditions, only a single peak was observed. N‐ and O‐glycans were chemically hydrolyzed by trifluoromethane sulfonic acid, resulting in a completely deglycosylated protein; alternatively, N‐glycans were enzymatically cleaved by incubation with peptide N‐glycosidase F (PNGase F). In both approaches a changed antibody pattern was detected, indicating that the separation is due to carbohydrate microheterogeneity of the protein. Deglycosylation of the antibody by treatment with PNGase F was investigated by matrix assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS). A shift to lower molecular masses of approximately 1500 Da for the enzymatically treated protein, compared with the intact glycoprotein, was found. The separation method was validated for linearity and reproducibility of migration time and peak area and optimized in terms of buffer pH, capillary temperature and borate concentration. This technique is sensitive to analyze batch‐to‐batch consistency in production and to test the stability of galenical formulations. After antibody storage in glass vials for 3 months at 37°C, the separation profile changed distinctly due to degradation at the carbohydrate or sialic acid moiety of the antibody, as indicated by MALDI‐TOF‐MS.
Detection and quantitation of specific mRNAs by ribonuclease protection assay using denaturing horizontal polyacrylamide gel electrophoresis: A radioactive and nonradioactive approach
Authors: Plath Annette , Peters Friedolf , Einspanier Ralf 
Published: May 2005

AbstractA radioactive (32P) and nonradioactive (digoxigenin) ribonuclease protection assay (RPA) has been developed to detect mRNAs of housekeeping proteins and growth factors. A modification of polyacrylamide gel electrophoresis (PAGE) to simplify RPA is described. Both Cleangels (Pharmacia) and laboratory‐cast polyacrylamide gels, in a denaturing, horizontal electrophoresis system, were used. The amount of toxic chemicals and waste was reduced, in comparison with sequencing gels normally used for RPA. The protected RNA fragments were shown to be well‐separated, with sufficient sensitivity in this modified, quick gel system.
Isoelectric focusing and titration curves in biomedicine and in agrofood industries: A multimedia teaching program
Authors: Cade‐Treyer Denise , Cade Ambroise , Darjo Alain , Jouvion‐Moreno Myrian 
Published: May 2005

AbstractThe aim of this 45 min, 60 megabyte, modular program is to initiate students, scientists and engineers of biotechnology, biomedicine and agrofood industries into isoelectric focusing (IEF) and titration curves for analytical (e.g. IEF, zone electrophoresis, isotachophoresis, electrotransfer) and preparative (e.g. ionexchange chromatography, chromatofocusing) application of charge‐dependent methods. For advanced teaching, the following theoretical and practical aspects may be of interest: pH gradient engineering, IEF resolving power, generation of pH gradient, sample‐ampholyte interactions, pH gradient drift, immobilized pH gradients (IPG), IPG‐two‐dimensional (2‐D) electrophoresis, preparative methods with multi‐compartments and IPG membranes, capillary IEF, isozyme analysis, etc.). The program associates fixed and animated drawings, and computer‐assisted simulations, with spoken and written commentaries (in English). It is illustrated with numerous IEF gel patterns and titration curves and some video sequences to be run on a multimedia PC with MS Windows 3.1 (or later releases) as the only software. The linear presentation of the program may be used directly on the PC, or may be projected on a screen from the PC, for small classes or for a larger audience (200 persons). Its development as an interactive multimedia program is in progress and will soon be available on the Internet.
Determination of the number of cysteine residues in high molecular weight subunits of wheat glutenin
Authors: Morel Marie‐Hélène , Bonicel Joëlle 
Published: May 2005

AbstractA simple method allowing the determination of the number of cysteine residues of the high molecular weight glutenin subunits (HMW‐GS) is presented. The method was adapted from that of Creighton (T. E. Creighton, Nature 1980, 2840, 487–489) with modified reagents for alkylation of the cysteine residues and the electrophoretic system. The acid‐urea‐polyacrylamide gel electrophoresis (PAGE) method, developed by Morel (M. H. Morel, Cereal Chem. 1994, 713, 238–242), was adopted and mixtures of iodoacetic acid and 4‐vinylpyridine were used to alkylate the glutenin subunits. The accuracy of the method was checked with some HMW‐GS whose number of cysteine residues was already known from molecular biology approaches. In subunits 5*, 2.2, 2.2* and 4.1 (of Dx types) from cultivars Ben and Fiorello, MG 7249, MG 315, and Kador, respectively, only 4 cysteine residues were demonstrated. Subunit 20 was found to exist as a regular Bx and By subunit pair, with the Bx subunit containing only 2 cysteine residues, which is unusual.
Characterization of natural peptides from bovine tissue using capillary electrophoresis, high performance liquid chromatography, matrix‐assisted laser desorption ionization, and Edman degradation
Authors: Weiler Angelika , Stoeva Stanka , Lehmann Rainer , Grübler Gerald , Paulus Gerd , Voelter Wolfgang 
Published: May 2005

AbstractA combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) spiking, Edman degradation, amino acid analysis and matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) was applied for the analysis of the primary structure of β‐thymosins. CE has been used to resolve peaks which coelute on HPLC, as well as to help establish the identity of tryptic fragments in the peptide mapping experiments of the two highly homologous β‐thymosins.
Electrophoretic and isoelectric focusing analysis of human recombinant alpha2‐HS glycoprotein produced in insect cells: Analysis of the post‐translational events
Authors: Kalabay Laszlo , Mathur Savita , Bobin Sophie , Arnaud Philippe 
Published: May 2005

AbstractAlpha2‐HS glycoprotein (AHSG) is a human serum glycoprotein synthesized by liver cells. It is a natural inhibitor of the insulin receptor tyrosine kinase activity. We produced this protein in insect cells by using a recombinant baculovirus expressing the whole coding sequence of the protein. By analyzing AHSG on isoelectric focusing and on sodium dodecyl sulfate (SDS) gels, followed by immunoblot, AHSG produced in insect cells was found to be phosphorylated and to possess the connecting peptide between the A and the B chains. The same features were found in the protein produced by Hep3B, a human liver cell line that synthesizes AHSG. By contrast, no phosphorylation could be detected in AHSG present in normal human plasma, and the connecting peptide was clipped. As the protein produced in insect cells is active on insulin receptors, in contrast to the plasma protein, our results suggest that the biological activity of the protein may be associated with its single chain form together with its phosphorylation.
Theoretical permutation gel electrophoretic analysis of a curved DNA fragment located in circular permutation
Authors: Nair T. Murlidharan , Madhusudan K. , Nagaraja V. , Kulkarni B. D. , Majumdar H. K. , Singh Rajan 
Published: May 2005

AbstractUsing the theoretical model for DNA curvature, we analyzed a set of fragments with a curved insert located in circular permutation. The theoretical permutation analysis of each of the cyclically located fragments reveals the presence of a shifting molecular bend locus. The delineation of the molecular bend locus associated with the fragments obtained by a second permutation helps in providing an explanation for the differential mobility behavior of the fragments.
Enhanced field strength and resolution in gel electrophoresis upon substitution of buffer by histidine at its isoelectric point
Authors: Chen Nong , Chrambach Andreas 
Published: May 2005

AbstractGel electrophoresis in isoelectric buffers, recently introduced by R. Westermeier and H. Schickle (Electrophoresis '95, Paris, Abstract No. 3, 1995), was applied to the automated HPGE‐1000 apparatus in the expectation to be able to increase the field strength under the limiting conditions of heat dissipation capacity and voltage of that apparatus. A previous attempt to achieve that aim by reduction of gel thickness had not yielded more than a twofold increment in resolving power. Replacing 0.2 × Tris‐boric acid‐EDTA (TBE) buffer, conventionally applied in the apparatus at 15 V/cm, by 0.05 M histidine, pH 7.6 (close to the pI of 7.47), allows one to increase the field strength to 60 V/cm, thus providing a nearly fivefold increment in resolution under otherwise identical conditions (fluorescein carboxylate‐labeled conalbumin‐sodium dodecyl sulfate (SDS) and soybean trypsin inhibitor‐SDS samples, 10°C, 4% MetaPhor agarose). An additional decrease in band dispersion can be obtained by decreasing the starting zone width through buffer dilution in the sample phase.