bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glutathione S‐transferases: Two‐dimensional electrophoretic protein markers of lead exposure
Authors: Witzmann Frank A. , Daggett Daniel A. , Fultz Carla D. , Nelson Shelli A. , Wright Lynda S. , Kornguth Steven E. , Siegel Frank L. 
Published: May 2005

AbstractGlutathione S‐transferases (GST) are a family of detoxification isoenzymes that catalyze the conjugation of xenobiotics and their metabolites with reduced glutathione. Lead exposure in rats is known to induce GST isoenzymes in the liver and kidney. These changes in expression have potential use as biomarkers of lead exposure. Because two‐dimensional electrophoresis (2‐DE) enables one to analyze both protein abundance changes and chemical changes in protein structure, 2‐DE was used to determine the effect of in vivo lead exposure on GST isoform expression in rat kidney cytosols. Male Sprague‐Dawley rats were exposed to inorganic lead, and proteins were separated by conventional ISO‐DALT and NEPHGE‐DALT techniques and blotted for immunological identification. Lead exposure caused detectable inductions in both GSTP1 and GSTM1 and quantifiable charge modification in GSTP1. These preliminary data confirm the utility of 2‐D electrophoretic GST analysis as indicative of lead exposure and toxicity and support its use for further elaboration of lead's effects on renal protein expression.
Incorporation of dispersion into the theory of electrophoresis of DNA and its complexes
Authors: Cann John R. 
Published: May 2005

AbstractIncorporation of the dispersion coefficient into the theory of the mobility‐shift assay for DNA‐protein complexes was highly successful largely due to increased mathematical rigor. A model simulating electrophoretic migration of DNA across the phase boundary between the initial zone of macromolecule and the gel lane predicts the peak asymmetry observed experimentally. It also predicts that, under the agency of the dispersion coefficient, the peak will become progressively more symmetrical during migration along the gel lane.
Rapid detection of γT cell receptor gene rearrangements in acute lymphoblastic leukemia by electrophoresis and silver staining: Implications for detection of minimal residual disease
Authors: Valetto Angelo , Lanciotti Marina , Martino Daniela Di , Anselmi Giorgia , Bottini Federico , Mori Piergiorgio , Candiano Giovanni 
Published: May 2005

AbstractMinimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) was studied using polymerase chain reaction (PCR). γT cell receptor (TCRG) genes are ideal targets for PCR‐based detection of MRD due to their molecular characteristics. Polyacrylamide gel electrophoresis (PAGE) analysis of PCR products followed by silver staining was performed for 72 children with ALL at the onset of disease. Silver staining is an effective technique to detect gene rearrangements without the use of ethidium bromide. Moreover, this method may show heteroduplex bands of a clonal nature when both TCRG alleles are rearranged. PCR products subjected to a rapid staining protocol were recovered from the gel, reamplified by a second PCR and directly sequenced. After sequencing, we identified the junctional region and obtained patient‐specific probes. In more than half of the patients we detected TCRG rearrangements that were used as molecular markers for residual disease.
Characterization of DNA standards by capillary electrophoresis
Authors: Atha Donald H. 
Published: May 2005

AbstractWe have used capillary electrophoresis to evaluate commercial DNA size standards and have found that it can provide an efficient assessment of size. However, the accuracy of the determination is adversely affected by anomalous migration times due to specific interactions of the DNA with the gel matrix as well as conformational differences in the DNA due to sequence heterogeneity. These anomalous migration times are strongly dependent on the choice of gel matrix. For example, the anomalous migration times that are observed in a 1 kilobase standard DNA ladder can be minimized using nongel hydroxyethylcellulose. In addition, the peak resolution can be increased and the anomalous migration can be reduced by the addition of the intercalating dye, ethidium bromide. However, in the case of the D1S80 allelic ladder, some of the DNA fragments possess nucleotide sequences which do not interact equivalently with the dye and produce irregular migration times. These measurements yield preliminary information useful in evaluating DNA size standards which may be used for a wide range of DNA diagnostic applications.
Apparent pore size of polyacrylamide gels: Comparison of gels cast and run in Tris‐acetate‐EDTA and Tris‐borate‐EDTA buffers
Authors: Stellwagen Nancy C. 
Published: May 2005

AbstractThe electrophoretic mobilities of DNA molecules in three different molecular weight ladders were measured in polyacrylamide gels containing different acrylamide concentrations (%T) and cross‐linker ratios (%C), cast and run in Trisacetate‐EDTA (TAE) buffer. The apparent pore radius of each gel was estimated from Ferguson plots of the relative mobiliteis of each of the DNA molecules, using the mobility of the monomer fragment in each molecular weight ladder as the reference mobility. The effective size of each of the DNA molecules was estimated from its radius of gyration. The apparent gel pore radii calculated in this manner ranged from 21 nm in gels containing 10.5 %T, 5%C to 200 nm in gels with 4.6%T, 2%C; similar to the values observed for polyacrylamide gels cast and run in Tris‐borate‐EDTA (TBE) buffer (Holmes and Stellwagen, Electrophoresis 1991, 12, 612–619). Hence, the effective pore size of polyacrylamide gels is essentially independent of whether the gels are cast and run in TAE or TBE buffer.
Isolation of ultrapure supercoiled plasmid‐DNA using preparative electrophoresis
Authors: Roeder Thomas , Görich Derk , Roeder Niklas P. , Bruchhaus Iris 
Published: May 2005

AbstractA large‐scale preparative polyacrylamide gel electrophoresis (PAGE) system for the isolation of high‐purity supercoiled plasmid‐DNA is described. This method should prove suitable for the isolation of large DNA molecules, either plasmid or linear DNA, that is required for the production of transgenic animals, for instance. The efficiency of the method is illustrated by the isolation of the gene for the green fluorescent protein, cloned into a mammalian expression vector and used for transfection of eukaryotic cells.
An apparatus for membrane‐confined analytical electrophoresis
Authors: Ridgeway Theresa M. , Hayes David B. , Moody Thomas P. , Wilson Timothy J. , Anderson Arthur L. , Levasseur John H. , Demaine Philip D. , Kenty Burt E. , Laue Thomas M. 
Published: May 2005

AbstractA membrane‐confined analytical electrophoresis apparatus for measuring the solution charge of macromolecules has been described previously (T. M. Laue et al., Anal. Biochem. 1989, 182, 377–382). Presented here is a design for this apparatus, which permits the on‐line acquisition and display of absorbance data from up to 512 positions along an analysis chamber. Concentration distributions of macromolecules in solution can be monitored in the chamber to provide steady‐state electrophoresis, electrophoretic mobility and diffusion measurements. Buffer chambers press semipermeable membranes against the open ends of a fused‐silica cuvette to form the analysis chamber. This configuration permits both the flow of buffer and the establishment of an electric field across the cuvette, while retaining macromolecules in the field of view. Though a gel may be included in the analysis chamber, none is required for gradient stabilization. The volume of sample required for analysis is 8 μL, most of which is recoverable. Experimental conditions can be varied during study by simply changing the circulating buffer and/or the electric field. The analysis and buffer chambers are held in an aluminum housing that sits in an aluminum water jacket. The water jacket provides temperature control, shielding from external electrical noise and also serves as an optical mask. Plans for the cell assembly, optical system and the computer interface for data acquisition are provided. The assembly and operation of the apparatus and the analysis of data are described.
Parallelism between width and asymmetry of peaks of rigid, spherical particles in capillary zone electrophoresis using polymer solutions
Authors: Radko Sergey P. , Chrambach Andreas 
Published: May 2005

AbstractPeak width and peak asymmetry of rigid spherical particles in the size range of 3–100 nm radius (R) were measured in capillary zone electrophoresis (CZE), using buffered uncross‐linked polyacrylamide of Mr 5.0 × 106. Polymer concentration‐dependent spreading of peak width and peak asymmetry were found to parallel one another. The parallelism holds whether the particle size is within the “small” (R < 20 nm) or “large” (R > 20 nm) size ranges previously found to differ in the mechanism of particle size dependent retardation of electrophoretic migration (S. P. Radko and A. Chrambach, Electrophoresis 1996, 17, 1094–1102). In application to the “small” particle size range, the parallelism between band width and band asymmetry can be qualitatively interpreted to be consistent with the Giddings‐Weiss mechanism (G. H. Weiss et al., Electrophoresis 1996, 17, 1325–1332) of electrophoresis in polymer‐containing media which postulates a dependence of band width and band asymmetry on the equilibrium between “stationary” and “mobile” states of the particle.
Ultrathin‐layer zone electrophoresis of alcohol dehydrogenase in partly rehydrated polyacrylamide gels: An alternative to starch gel electrophoresis
Authors: Maly I. Piotr , Toranelli Mireille , Crotet Valérie , Sasse Dieter 
Published: May 2005

AbstractA highly sensitive electrophoretic technique for the separation of alcohol dehydrogenase isoenzymes by zone electrophoresis in partly rehydrated polyacrylamide gels is described. Five hundred μm thin polyacrylamide gels are polymerized under standardized conditions. After polymerization the gels are washed thoroughly with distilled water to remove any unreacted monomers, catalysts or still soluble polymers. The washed gels are then impregnated with 0.5% Tween 20 and dried. Before electrophoresis the dry gels are rehydrated to a thickness of 250 μm, which makes up 50% of the original gel volume. Rehydration is carried out by use of a degassed buffer solution. This method permits the demonstration of the isoenzymes of alcohol‐dehydrogenase class I and II in man and allows quantitative determination.
Surface modification based on Si‐O and Si‐C sublayers and a series of N‐substituted acrylamide top‐layers for capillary electrophoresis
Authors: Gelfi Cecilia , Curcio Mario , Righetti Pier Giorgio , Sebastiano Roberto , Citterio Attilio , Ahmadzadeh Hossein , Dovichi Norman J. 
Published: May 2005

AbstractTwo approaches were used to prepare a series of surface‐modified capillaries. In the first, a sublayer was formed by coupling γ‐methacryloxypropyltrimeth‐oxysilane to the surface silanol groups forming an SI‐O bond; a top layer was then formed by polymerizing acrylamide in the capillary, which reacted with the sublayer. In the second approach, a sublayer was formed by silanol chlorination, followed by Grignard coupling of vinylmagnesium bromide to form an Si‐C bond at the surface; a top layer was formed by polymerizing either acrylamide (AA), dimethylacrylamide (DMA), N‐acryloylaminoethoxyethanol (AAEE), or N‐acryloylaminopropanol (AAP) onto the sublayer. The Si‐C‐poly(AA) capillaries were more stable and produced an approximately 10‐fold lower electroosmotic flow compared to the Si‐O‐poly(AA) capillaries. The Si‐C sublayer was used to compare the performance of all four top layers. Electroosmotic flow decreased in the order: Si‐O‐poly(AA), Si‐C‐poly(AA), Si‐C‐poly(AAEE), Si‐C‐poly(DMA), and Si‐C‐poly(AAP). Si‐C‐poly(AA) showed evidence of irreversible degradation at pH 9 already after 40–50 runs. Si‐C‐polyAAP‐coated capillaries demonstrated superior efficiency and migration time reproducibility for a number of alkaline proteins and for fluorescently labeled ovalbumin. Excellent performance was maintained, in the case of poly(AAP), for a least 300 runs (of 30 min duration) at pH 9.0.
Predictive model for capillary electrophoretic peptide mobility in 2,2,2‐trifluoroethanol‐water solution
Authors: Castagnola Massimo , Rossetti Diana Valeria , Corda Marcella , Pellegrini Mariagiuseppina , Misiti Francesco , Olianas Alessandra , Giardina Bruno , Messana Irene 
Published: May 2005

AbstractUsing capillary electrophoresis (CE) on a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da, the Stokes radii at different protonation stages and the acidic dissociation constants in water and in a 2,2,2‐trifluoroethanol (TFE) water mixture (30% v/v) were determined. These results permitted us to establish separately the reliability of semiempirical models utilized for the prediction of peptide size and charge at different acidic pHapp (pHapp range: 2.00–4.25). The data obtained on size and charge were utilized in order to provide suitable mobility predictions on the basis of the charge‐to‐size ratio. The best predictive conditions for size and charge were found at the most acidic range of pHapp studied (2.00–2.25), either in water or a TFE‐water mixture, and reliable predictive equations for peptide mobility were established at this pHapp.
Electrophoretic analyses in a case of monoclonal γ chain disease
Authors: Tissot Jean‐Daniel , Tridon Arlette , Ruivard Marc , Layer Andreas , Henry Hugues , Philippe Pierre , Schneider Philippe 
Published: May 2005

AbstractAbnormal low molecular weight immunoglobulin heavy chain can be detected in serum samples from patients with heavy chain disease. In this paper, we report the characterization of a γ heavy chain in a patient suffering from a lymphoplasmocytic disorder, using immunofixation analysis and two‐dimensional polyacrylamide gel electrophoresis. We demonstrate that the combination of serum protein agarose electrophoresis and two‐dimensional electrophoresis (three‐dimensional electrophoresis) can be used to further characterize abnormal protein bands detected by immunofixation.
Isoelectric focusing in the vegetable seed industry
Authors: Van Den Berg Bartel M. 
Published: May 2005

AbstractDifferent types of electrophoretic procedures were developed for hybrid purity testing based on starch gel electrophoresis (SGE), vertical polyacrylamide gel electrophoresis (PAGE), and isoelectric focusing (IEF). For the most important vegetables these methods are much faster than plant grow‐outs and relatively inexpensive. Compared to SGE and PAGE methods, horizontal IEF proved to be more efficient for large‐scale hybrid purity testing. These developments were made possible by the basic work of Harry Rilbe and improvements that were initiated as a result of Rilbe's work. The present paper describes a number of milestones during this developmental period, starting with the isoelectric focusing concept of Harry Rilbe up to the large‐scale application of IEF. Further, a comparison of IEF with DNA fingerprinting methods along with the future of both techniques is discussed with respect to hybrid purity testing in the vegetable seed industry. When it comes to a choice between the use of either IEF or a DNA‐based method, efficiency and efficacy determines the method which is best suited for hybrid purity testing. It is also concluded that in the future we will see an increased use of both IEF as well as DNA‐based methods for hybrid purity testing because expectations of growers has increased; consequently they will accept fewer inbreds in a hybrid variety, especially when growing in a greenhouse.
Detection of enzymes active on various β‐1, 3‐glucans after denaturing polyacrylamide gel electrophoresis
Authors: Trudel Jean , Grenier Jean , Asselin Alain 
Published: May 2005

AbstractEnzymes were assayed for glucanase activity after denaturing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in gels containing β‐1,3‐glucans embedded as substrate. Lentinan, curdlan, paramylon, baker's yeast alkali‐insoluble glucan, baker's yeast alkali‐soluble glucan and carboxymethyl (CM)‐pachyman were compared to oligomeric laminarin, which is the usual substrate for assaying β‐1,3‐glucanase activities. Detecting enzyme activities by aniline blue fluorescent staining was also compared with the staining of released reducing sugars by 2,3,5‐triphenyltetrazolium chloride (TTC). For the nonreduced proteins, the Driselase extract exhibited one major band at 32.5 kDa and one less intense band at 23 kDa for most substrates with the two detection procedures. No Lyticase enzyme was detected in either detection procedures for all tested substrates. For barley enzymes, no activity was revealed after aniline blue staining while one undescribed 19 kDa glucanase activity was best shown after TTC staining with curdlan, paramylon and CM‐pachyman as substrates. In the case of reduced proteins, the Lyticase extract yielded three bands (33, 36 and 46 kDa) on several substrates with both detection procedures. This was the same for the barley leaf extract (32, 36 and 39 kDa). The Driselase extract showed one 42 kDa band. Many enzymes active on β‐1,3‐glucans are thus best revealed when proteins are denatured and reduced and when protein renaturation after SDS‐PAGE involves a pH 8.0 treatment and the inclusion of 1 mM cysteine in buffers. However, some enzymes are only detected when proteins are denatured without reduction. Finally, the use of various polymeric β‐1,3‐glucan substrates different from oligomeric laminarin is necessary to detect new types of enzymes such as the 19 kDa barley glucanase.
Expression of recombinant psoriasis‐associated fatty acid binding protein in Escherichia coli: Gel electrophoretic characterization, analysis of binding properties and comparison with human serum albumin
Authors: Vorum Henrik , Madsen Peder , Svendsen Ib , Celis Julio E. , Honoré Bent 
Published: May 2005

AbstractThe psoriasis‐associated fatty acid binding protein (PA‐FABP, also known as FABP5) is a novel keratinocyte protein that is highly up‐regulated in psoriatic plaques (P. Madsen, H. H. Rasmussen, H. Leffers, B. Honoré and J. E. Celis, J. Invest. Dermatol. 1992, 99, 299–305). Here we have expressed PA‐FABP in Escherichia coli as a fusion protein containing an NH2‐terminal hexa‐His tag followed by a factor Xa cleavage site. The recombinant protein was expressed at a level of about 30% of the soluble proteins and was purified to homogeneity using a simple two‐step protocol consisting of affinity chromatography on Ni2+‐nitrilotriacetic acid agarose followed by gel filtration. The recombinant protein was then digested with factor Xa and characterized by two‐dimensional gel electrophoresis. The ability of PA‐FABP to bind saturated fatty acids ranging from 6 to 16 carbons was determined directly by dialysis and compared to human serum albumin (HSA). The results showed that PA‐FABP binds multiple molecules of the fatty acids hexanoate (C6:0), octanoate (C8:0), decanoate (C10:0) and laurate (C12:0) with a K1 of about 104 m−1, and myristate (C14:0) with a K1 of 4.4 × 105 m−1. Palmitate (C16:0) also bound strongly with multiple molecules. Due to the very low solubility of palmitate its affinity to PA‐FABP was measured relatively to HSA and found to be 8.1 times lower. At ligand/protein ratios below 1, all fatty acids bound to PA‐FABP with about one to three orders of magnitude lower affinity than to HSA. The difference in the fatty acid binding properties of the two proteins may reflect differences in their three‐dimensional structures, which in the case of PA‐FABP consists mainly of β‐sheets while HSA contains predominantly α‐helices.
Rab 11a is modifiedin vivoby isoprenoid geranylgeranyl
Authors: Gromov Pavel , Celis Julio E. 
Published: May 2005

AbstractPost‐translational adduction of farnesyl or geranylgeranyl moieties to a terminal cysteine residue of proteins is a characteristic feature ofras‐related GTP‐binding proteins. According to current rules of prenylation the carboxylterminal motif (CXXX or CC/CXC) as well the context of the cysteine residue dictate the extend and specificity of the isoprenoid modification. Rab 11a, a small GTP‐binding protein that is associated with pathways regulating protein traffic, terminates with isoleucine at itsC‐terminus, suggesting that it may only be geranylgeranylated. Recent finding, however, showed that rab 11a can be modifiedin vitroby different prenyl groups: farnesyl and geranylgeranyl [1]. To determine whether rab 11a is modifiedin vivoby both isoprenoids we transiently overexpressed rab 11a in COS1 cells, and analyzed the translation products by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) in combination with metabolic labeling in the presence of either [3H]farnesyl‐PP or [3H]geranylgeranyl‐PP. Contrary to thein vitroresults, our studies showed that rab 11a is post‐translationally modifiedin vivoonly by geranylgeranyl isoprenoid. The data implied thatin vivothere must exist other determinant(s) that are necessary for prenyltransferase recognition.
Rapid isolation of phage displayed antibodies to β‐actin eluted from two‐dimensional electrophoresis gel
Authors: Zhou Jian‐Nian , Linder Stig , Franzén Bo , Auer Gert , Hochstrasser Denis F. , Persson Mats A. A. 
Published: May 2005

AbstractWe describe a simple and efficient procedure which can be used to prepare antibodies to proteins extracted by two‐dimensional gel electrophoresis (2‐DE), using β‐actin as a model. Protein was electroeluted from a stained gel, biotinylated and used for selection of phage from a semisynthetic phage antibody library. After four rounds of selection using 50 ng β‐actin per cycle, approximately 8 × 103 phage were recovered. Antibody fragments were prepared from 21 randomly picked clones. Six of eighteen (6/18) antibody‐positive clones produced antibody fragments reacting against β‐actin in an enzyme linked immunosorbent assay (ELISA). Sequencing of the HC‐CDR3‐region showed that all six clones were independent isolates, suggesting that a large number of independent phage antibody reactivities were generated.
Analysis of Bence Jones proteinuria by high resolution two‐dimensional electrophoresis
Authors: Williams Katherine M. , Williams John , Marshall Thomas 
Published: May 2005

AbstractAnalysis of Bence Jones proteinuria by high resolution two‐dimensional electrophoresis (2‐DE) and immunoblotting reveals a complex pattern of light chain (LC) isoforms corresponding to the free monoclonal Bence Jones protein and its fragments. Replica blotting gives duplicate blots for LC typing (λ, ϰ) and, under the conditions employed, leaves sufficient protein for Coomassie Blue staining of the urinary protein profile and pI/Mr determination of the LC isoforms. Carrier ampholytes (CAs, in our “simplified” 2‐DE system) and immobilised pH gradients (IPGs, in the Multiphor 2‐DE system) give similar LC isoform patterns. Artifacts, including cone‐like distortions and trailing “piggyback” spots, are visualised with both 2‐DE systems. IPGs are advantageous as they allow reproducible detection of strongly basic LC isoforms by isoelectric focusing (under equilibrium conditions) without recourse to CA nonequilibrium pH gradient electrophoresis.