bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two–dimensional polyacrylamide gel electrophoresis, with immobilized pH gradients in the first dimension, of barley seed proteins: Discrimination of cultivars with different malting grades
Authors: Görg Angelika , Postel Wilhelm , Baumer Max , Weiss Walter 
Published: May 2005

AbstractThe suitability of high‐resolution two‐dimensional gel electrophoresis for barley cultivar discrimination and for classification with respect to their malting properties was studied. Seed proteins of 14 barley cultivars with different malting qualities were extracted with urea/dithiothreitol/Nonidet P‐40 buffer and subjected to two‐dimensional gel electrophoresis with immobilized pH gradients in the first dimension (IPG‐DALT). The results of IPG‐DALT were compared to the protein patterns obtained by a standard technique, sodium dodecyl sulfate polyacrylamide gel electrophoresis of hordeins. Sodium dodecyl sulfate‐gel electrophoresis yielded seven different “B” and four different “C” hordein patterns; “A” and “D” hordein patterns were uniform in all cultivars tested. Four cultivars could be distinguished unequivocally, the others were classified into three groups containing between two and five cultivars. In contrast to these findings, IPG‐DALT yielded three different “A”, eight different “B”, four different “C” and two different “D” hordein patterns. When the “A”, “B”, “C” and “D” hordein patterns were combined, ten cultivars exhibited unique hordein patterns whereas the remaining ones were classified into two groups containing two cultivars each. Moreover, when albumin and globulin proteins were used for evaluation in addition to the hordeins, all cultivars could be discriminated by IPG‐DALT. IPG‐DALT, performed on small‐scale and/or ready‐made gels, proved to be an ideal complementary system to one‐dimensional electrophoretic methods for routine seed testing purposes because of its speed, reliability, and simplicity. IPG‐DALT was also applied to study the relationship between the different polypeptide patterns and the malting quality. Although cultivars with identical one‐dimensional protein patterns but different malting quality could be successfully differentiated by IPG‐DALT, a direct correlation between specific protein spots or protein patterns to the malting quality was not found within the cultivars tested.
Monitoring by two‐dimensional electrophoresis somatic embryogenesis in leaf and petiole explants from Vitis
Authors: Gianazza Elisabetta , De Ponti Paola , Scienza Attilio , Villa Pierluigi , Martinelli Lucia 
Published: May 2005

AbstractA two‐dimensional electrophoretic analysis of the total proteins was carried out in Vitis rupestris as model system in order to characterize the different developmental stages – from callus to plantlets – of somatic embryogenesis events in the grapevine. The patterns of callus, embryogenetic callus, somatic embryos and plantlets derived from leaf and petiole explants were compared. Each differentiation step was characterized by specific peptide spots.
The use of immobilized pH gradients for the detection of human polymorphisms in the forensic identification of bloodstains
Authors: Muñoz‐Barús Ignacio , Carracedo Angel , Pascali Vincenzo L. 
Published: May 2005

AbstractSome polymorphic proteins (α1‐antitrypsin, orosomucoid, transferrin, group specific component, plasminogen) and enzymes (phosphoglucomutase, acid phosphatase, estrase D) were determined in bloodstain extracts by isoelectric focusing with carrier ampholytes (CA) and with immobilized pH gradients (IPGs) rehydrated with CA. IPGs yield superior results for typing of genetics markers in bloodstains since phenotypes are better distinguished and the bands are straighter and sharper in the presence of contaminants. Also, the sensitivity of IPGs with CA is similar to isoelectric focusing (IEF) with CA. A new variant, ACP*B1, found in Negroid west African populations and not found in Caucasians is described. Such a variant can only be determined by IPGs since its isoelectric point (pI 5.95) is close to that of the ACP*B (pI 6.05) variant.
Rapid and simple method for the identification of apolipoprotein E isomorphic phenotypes from whole serum
Authors: Kohlmeier Martin , Drossel Hans‐Joachim , Sinha Pranav , Köttgen Eckart 
Published: May 2005

AbstractFor the identification of apolipoprotein E isomorphic phenotypes, fresh or thawed serum was analyzed without prior delipidation or other pretreatment. Using 5% polyacrylamide gels with a 40 mm interelectrode distance, the isoforms were separated by isoelectric focusing in immobilized pH gradients ranging from pH 5 to 6.5, and transferred onto polyvinylidene difluoride membranes by contact blotting for 1 h. The apolipoprotein E isoforms were identified following immunostaining. The electrophoresis required less than 2 h and the entire procedure could be completed within 6 h.
Microheterogeneity of apolipoprotein D as revealed by electroblotting following isoelectric focusing in Immobiline DryPlates
Authors: Holmquist Leif 
Published: May 2005

AbstractThe microheterogeneity of apolipoprotein D was examined by a procedure involving, in sequences: (i) electrophoresis in an immobilized pH 4–7 gradient in an Immobiline DryPlate‐polyacrylamide gel supplemented with Ampholine pH 5–7, (ii) covering of the gel with sodium dodecyl sulfate‐containing agarose, (iii) electroblotting onto a polyvinylidene difluoride membrane and (iv) immunological identification. Seven isoforms were obtained with partially purified apolipoprotein D. Using this technique the apparent pI values at 15°C for the isoforms were 4.57, 4.67, 4.78, 4.83 and 5.95, 6.06 and 6.19 (SD ± 0.05 for all). Direct staining of the Immobiline DryPlate could not reveal the isoforms of partially purified apolipoprotein D.
Modified silver staining for immobilized pH gradients
Authors: Rabilloud Thierry , Brodard Virginie , Peltre Gabriel , Righetti Pier Giorgio , Ettori Claudia 
Published: May 2005

AbstractSilver development of gels containing an immobilized pH gradient has proved difficult so far because the bonded buffers (especially the tertiary amino acrylamido derivatives) tend to adsorb silver ions with a resultant heavy background of increasing darkness from the anode to the cathode. We report a variant of silver staining in which thiosulfate is used twice: (i) prior to silver impregnation, at the millimolar level, to enhance sensitivity, and (ii) during development, at the micro‐molar level, to decrease the background.
High resolution two‐dimensional gel analysis of proteins in the central nervous system of larvae of Drosophila melanogaster
Authors: Assigeo Rosa , Santarén Juan F. 
Published: May 2005

AbstractAn improved method of high resolution two‐dimensional gel electrophoresis was used to study the patterns of protein synthesis in the central nervous system (CNS) of late instar larvae of Drosophila melanogaster. A small number of CNSs was radiolabeled with a mixture of 14C‐labeled amino acids or with [35S]methionine, and the pattern of labeled proteins was analyzed. One thousand and forty‐five polypeptides, 800 acidic (IEF) and 245 basic (NEPHGE), from the CNS of several wild‐type strains have so far been separated and cataloged. All these polypeptides were numbered and compared with our catalog of polypeptides from wing imaginal discs, and quantitative or qualitative changes were detected in more than 23% of the polypeptides. When comparing patterns of label of CNSs we found small quantitative differences in the rate of synthesis between individuals of the same strain, not due to sexual differences, and a minute number of quantitative and qualitative differences between groups of individuals of different strains.
Errors in sizing bands of hypervariable DNA profiles on autoradiograms: Are they Gaussian?
Authors: Pascali Vincenzo L. , Moscetti Alessandra , Dobosz Marina , Pescarmona Marina , d'Aloja Ernesto 
Published: May 2005

AbstractThe accuracy of procedures for sizing hypervariable restriction fragments by Southern blot analysis (SBA) has been tested under three different experimental conditions: (i) intrablot serial analyses: three heterozygous DNA profiles were tested 14 times each in the same gel electrophoresis; (ii) intralaboratory analyses: we replicated three profiles (six autoradiographic bands) in over 100 SBA experiments; (iii) interlaboratory analyses: 15 serial measurements produced in a recent collaborative study (Forensic Sci. Int. 1991, 49, 1–15) were taken into account. In these three cases, a typical U‐shaped correlation curve between molecular size and coefficient of variation was found. We explain decrease of accuracy at both extremities of the gels in terms of: (i) enlarged shapes of bands and mean electrophoretic resolution at the cathode; (ii) diffusion and blurring of bands at the anodal edge. The three populations of data were subjected to a χ2 test and to the Kolmogorov‐Smirnov test in‐order to verify their compliance with normal distribution. Twenty‐three out of 27 tests indicated no significant deviation from the assumption of Gaussian distribution. We recommend the adoption of tests for normality to validate the use of symmetric confidence intervals for calculating gene frequencies and asserting a match between adjacent bands.
Analysis of collagen type III by uninterrupted sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblotting: Changes in collagen type III polymorphism in aging rats
Authors: Takasago Teiichi , Nakamura Kazuyuki , Kashiwagi Shiro , Inoue Shin‐Ichi , Ito Haruhide , Takeo Kazusuke 
Published: May 2005

AbstractA new method of type III collagen analysis by uninterrupted sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) combined with immunoblotting was developed. The electrophoresis was carried out with gels containing 4 M urea. A negatively charged reducing agent, thioglycolic acid, was added to the running buffer of the cathodic reservoir between 15 and 20 min after Bromphenol Blue (BPB) migrated to the top of the separating gel, to reduce interchain disulfide binding of the collagen. The polymorphic type III collagens, i.e., an α‐chain derived from a trimer [α1(III)]3 with interchain disulfide bonds but without covalent cross‐links, α1(III), a β‐chain with covalent cross‐links, β(III), or an α‐chain released from a trimer without reduction of the disulfide bonds, α*1(III), were identified by immunostaining and quantified by densitometry. Using this method, changes in collagen type III polymorphism with aging were examined in the aorta, brachial artery, and skin of rats. The total quantity of collagen type III decreased with aging in all tissues. β(III) was the major component in the aorta and brachial artery, but α1(III) was the major component in the skin. With increasing age from 3 to 60 weeks, the ratio of β(III) to α1(III), which is correlated with the extent of covalent cross‐linking, showed a steep increase in the aorta but only a slight increase in the skin and it remained almost constant in the brachial artery. The ratio of α*1(III) to α1(III), which is correlated with the extent of degradation, increased steeply from 12 weeks to 60 weeks in the aorta; in the brachial artery and the skin, however, it increased up to the 22nd week and was, thereafter, followed by almost constant values. These findings are characteristic of the profile of collagen type III metabolism in each of these tissues with aging.
Analysis of human tear proteins by two‐dimensional electrophoresis
Authors: Mii Seijiro , Nakamura Kazuyuki , Takeo Kazusuke , Kurimoto Shinji 
Published: May 2005

AbstractHuman tear proteins in the conjunctival sac were separated on the basis of the differences in their isoelectric points and molecular weights using micro two‐dimensional electrophoresis combined with immunoblotting. The two‐dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis were compared with those from normal individuals. We also measured integrated intensities of seven protein spots, lactoferrin (LF), albumin and five specific tear proteins (STP), to examine differences in the amounts of these proteins in tears from normal individuals of different sexes. In the tears from patients with conjunctivitis, secretory immunoglobulin A (IgA), LF and STP spots were stained more weakly, whereas the albumin spot was stained more strongly as compared with those from normal individuals. Furthermore, haptoglobin and IgG spots appeared in the tears from patients with conjunctivitis. These were more prominent in the tears from patients with severe conjunctivitis. There were significant differences in the amounts of LF and two kinds of STPs in the different sexes. The amounts of these proteins were larger in females.
Purification of serum amyloid A and its isoforms from human plasma by hydrophobic interaction chromatography and preparative isoelectric focusing
Authors: Malle Ernst , Heß Horst , Münscher Gerhard , Knipping Gabriele , Steinmetz Armin 
Published: May 2005

AbstractThe present work was aimed at isolating human serum amyloid A, (SAA), an acute‐phase protein mainly complexed to high density lipoproteins, directly from human plasma without sequential ultracentrifugation of lipoproteins and subsequent delipidation of the apolipoprotein moiety. Hydrophobic‐interaction fast‐protein liquid chromatography on Octylsepharose, using stepwise gradient elution profiles under dissociating conditions, followed by fast‐protein liquid‐gel permeation chromatography on a Superdex TM75 column revealed a higher than 95% purity of isolated SAA. Further purification of SAA from coeluting apolipoproteins C and A‐II was achieved by preparative isoelectric focusing between pH 5–7 using a Rotofor apparatus. Separation of the main SAA isoforms, SAA1 (pI 6.5) and SAA1 des‐Arg (pI 6.0, lacking the N‐terminal arginine), was achieved by anion‐exchange fast‐protein liquid chromatography on a Fractogel EMD DEAE 650‐S column. The purity of the SAA1 and SAA1 des‐Arg isoforms, thus isolated, was checked by immunochemical techniques and amino acid analysis. With the described method various SAA isoforms can be isolated, purified and separated directly from human plasma/serum without prior ultracentrifugation.
A comparison between low background silver diammine and silver nitrate protein stains
Authors: Rabilloud Thierry 
Published: May 2005

AbstractSeveral methods of silver staining of proteins after sodium dodecyl sulfate‐electrophoresis in polyacrylamide gels were compared. The most rapid methods were found to be less sensitive than more time‐consuming methods. Among the long methods, those using glutaraldehyde treatment of the gel and silver diammine complex as the silvering agent were found to be the most sensitive, at the expense of the use of a modified polyacrylamide matrix and higher silver concentrations.
Theory of band broadening for DNA gel electrophoresis and sequencing
Authors: Slater Gary W. 
Published: May 2005

AbstractThe problem of (thermal) band broadening during DNA gel electrophoresis is studied analytically and numerically using the reptation model. It is shown that the orientation of the end‐to‐end vector of the molecules leads to increased diffusion and even to molecular size‐independent band broadening. Thus, the Einstein relation between mobility and diffusion holds for only very small molecules. Also, “self‐trapped” molecules are predicted to give fuzzy bands, as observed experimentally. Finally, megabase molecules are discussed and the consequences of our predictions for the improvement of sequencing are considered.
Gel electrophoresis of an end‐labeled DNA I. Dynamics and trapping in constant fields
Authors: Défontaines Anne‐Dominique , Viovy Jean‐Louis 
Published: May 2005

AbstractA theory for the gel electrophoresis of a flexible polyelectrolyte, bearing an uncharged bulky label or an uncharged section at one end, is presented. We first consider a gel that is fully permeable to the label: we calculate the degree of stretching of the polyelectrolyte and its mobility as a function of chain size, electric field and label friction. Various regimes are identified, and their “existence domains” are calculated. For increasing friction, we predict a transition from a mobility decreasing with chain size to a mobility increasing with chain size. Secondly, we consider the possibility that the label may get trapped at some locations of the gel, a situation relevant to a method of “trapping electrophoresis” recently proposed by Ulanovsky et al. for DNA sequencing. A molecular model for detrapping by thermally activated “backward reptation” is constructed and solved using the Kramers rateequation theory. Different closed analytical expressions and approximate scaling laws corresponding to different regimes of stretching and field strengths are predicted. The most striking result is a mobility which exponentially decreases past a critical size N, which decreases with increasing field. In the regime relevant to the experiments by Ulanowsky et al., we predict N ∼ E−2/3. The predictions are in good qualitative agreement with presently available experiments, but further experimental investigations are suggested.
Effects of anomalous migration and DNA to protein ratios on resolution of equilibrium constants from gel mobility‐shift assays
Authors: Senear Donald F. , Dalma‐Weiszhausz Dennise D. , Brenowitz Michael 
Published: May 2005

AbstractNumerical resolution of binding constants from data generated by the gel mobility‐shift assay is dependent on the experimental resolution of the different ligation states of the DNA. Previously we showed that the populations of the intermediate ligation states at partial saturation with the protein ligand are extremely sensitive to cooperativity (Senear, D. F. and Brenowitz, M. J. Biol. Chem. 1991, 266, 13661–13671). This makes accurate gel mobility‐shift data extremely useful to the demonstration of cooperativity. However, the accuracy with which the intermediate ligation state populations are resolved has been questioned. Thus, two additional and related questions are now considered. First, what information is available if the intermediate ligation state populations are not used in the analysis of binding constants. Second, is accurate information obtained from those states under conditions of high DNA concentration. These questions are addressed by using the interactions of the λ cI repressor protein with the three site operator, OR, and the interaction of the E. coli GalR protein with the single site operator, OE. Both simulated and experimental data are analyzed. The results point to two conclusions. First, precise resolution of all macroscopic constants for binding of proteins to DNA is critically dependent on the intermediate ligation state populations; resolution is limited to at most two DNA sites if these states are not used in the analysis. Second, when the DNA and protein concentrations used in the titrations are comparable, the resolution of binding constants is extremely sensitive to experimental uncertainty in the macromolecule concentrations.
Electroblotting of proteins on agarose gel containing specific antibodies
Authors: Atmeh Ragheb F. , Abuharfeil Nizar M. 
Published: May 2005

AbstractA method is described for electroblotting of proteins, separated by gradient polyacrylamide gel electrophoresis, onto an agarose gel matrix containing specific antibodies. Three proteins of different molecular weight, including human albumin, isolated and in plasma, human plasma transferrin and C3 complement were tested. Immunoblotting on agarose, compared with nitrocellulose, was quantitative and highly sensitive, with small amounts of protein (i.e., 100 pg) being detected. Moreover, albumin aggregates (i.e., dimer, trimer and tetramer) were blotted quantitatively in addition to the monomer, and their percentages were calculated. This method is sensitive, quantitative, reproducible, and includes fewer manipulations; furthermore, it is less expensive and does not require the use of toxic or carcinogenic agents.
Lane distortions in gel electrophoresis patterns
Authors: Starita‐Geribaldi Mireille , Houri André , Sudaka Pierre 
Published: May 2005

AbstractDistortions effects were studied as a function of gel environment, apparatus design and buffer type. Outward lanes distortions pronounced in low conductivity buffers for both continuous buffer systems and stacking gels, were field strength dependent. In discontinuous buffer systems, the moving boundary of the Laemmli buffer system deformed depending on the environment. In the Michov buffer system, the high conductivity resolving gel surrounded by a low conductivity electrode buffer, displayed straight pathways until a field strength of 15 V/cm, permitting to obtain information about lanes distortions depending on the acrylamide matrix structure. A decreased ammonium persulfate concentration, e.g. to 0.03%, induced phase segregation in gels at low temperature during the run: a critical endpoint near 11°C for a gel in the Michov buffer system under conditions of electrophoresis was supposed. Gel collapse resulted in inward deviations in the lanes of the pattern and also faint bands in the zones where the gel was retracted. Besides this effect of catalyst concentration, the inwardly distorted pattern depended on temperature, field strength, solvent and hydrolysis due to gel aging. Amendments for overcoming lane distortions are suggested.
Electrotransfer of fixed phosphoproteins from pieces of dried polyacrylamide gel to small disks of nitrocellulose, nylon or polyvinylidene difluoride
Authors: Gonçalyes Carlos A. , Rodnight Richard 
Published: May 2005

AbstractA simple method for the transfer of 32P‐labeled proteins from dried polyacrylamide gels to small disks of nitrocellulose, nylon or polyvinylidene difluoride (PVDF) is described. Gel pieces containing the desired phosphoprotein are rehydrated in buffer containing sodium dodecyl sulfate (SDS) and sealed in agarose in a glass tube over a supporting gel of polyacrylamide. Protein is transferred upwards through a discontinuous density gradient of SDS‐buffer and methanol to a disk of membrane sealed to the mouth of the tube with dialysis membrane. The method allows the concentration of a phosphoprotein present in several gel pieces to a single disk of immobilized membrane. Recovery of phosphoprotein was at least as good as obtained with conventional electroblotting. Application of the method to the analysis of the phosphoamino acid content of the astrocyte marker, glial fibrillary acidic protein, is described.
Differentiation of proteins in polyacrylamide gels by a modification of silver staining for the PhastSystem and a laser densitometer
Authors: Kierdorf Horst , Melzer Heinrich , Mann Helmut , Sieberth Heinz‐Günther 
Published: May 2005

AbstractNonspecific background staining of the gel matrix is the limiting factor in the differentiation of proteins using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. It is caused by nonspecific binding of silver ions in the gel matrix, either to nonprotein compounds or through the chemistry of the polyacrylamide gel itself. The resulting stain of the gel produces a stain baseline, making differentiation of protein bands with a laser densitometer difficult. We have therefore developed a modified silver staining method for the PhastSystem to reduce such nonspecific background staining. Apart from slowing the development program by lowering the temperature to 15°C and shortening the incubation time to 4 min, the essential step in the modification is the combined use of an EDTA and Tris‐acetate buffer solution which stops the reduction of silver ions. The reduced background staining leads to an improved detection of protein bands and virtually identical zero lines for the laser densitograms and the stain baselines. The total staining time is 91 min. All steps in the program are fully automated and continuous, employing the PhastSystem staining unit.