bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alcian blue fixation allows silver staining of the isolated polysaccharide component of bacterial lipopolysaccharides in polyacrylamide gels
Authors: Corzo Javier , Pérez‐Galdona Ricardo , León‐Barrios Milagros , Gutiérrez‐Navarro Angel M. 
Published: May 2005

AbstractThe effect of the cationic dye Alcian Blue on the silver staining of bacterial lipopolysaccharide and its polysaccharide and lipid A portions in polyacrylamide gels was investigated. The polysaccharide was only stained when the gel was previously treated with the dye. The polysaccharide moiety was found to be responsible for the lipopolysaccharide staining with silver, whereas the lipid A seemed unimportant. Treatment with Alcian Blue may prove useful to detect hydrophilic components of lipopolysaccharide samples that could no be stained by the usual silver staining procedures.
An improved method combining two electrophoretic procedures: Application to the separation of lens α‐crystallin isoforms
Authors: Arruti Cristina , Chifflet Silvia 
Published: May 2005

AbstractPolypeptides having different net electric charges and very similar molecular weights, visualized as one single band in sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS‐PAGE), can be readily analyzed by an improved method combining two electrophoretic procedures. The methodology consists of the identification and isolation of selected protein bands from SDS‐PAGE, their equilibration in an isoelectric focusing (IEF) sample buffer, and their casting and separation in an IEF flat‐bed gel. This method requires no extra equipment, is highly reproducible, is suitable for quantitative and comparative studies, and is especially useful in the case of small samples. As a particular example, we analyze here the subunit composition of α‐crystallins of young and embryonic quail lenses.
Electrophoresis with intermittent scanning of the migration path: Detection of resolution within shortened time
Authors: Deml Mirko , Garner Mark M. , Chrambach Andreas 
Published: May 2005

AbstractIntermittent optical scanning (by detection of optical density or fluorescence) of the electrophoretic migration path was applied to the resolution of two dyes under an arbitrary set of conditions. Scanning at 5‐min intervals allows for detection of resolution between the two zones at least 3 times faster than conventional automatic zone detection employing a detector at the end of the migration path. This result promises that replacing stationary by mobile detectors in general would result in a substantial time saving for automated detection of electrophoretic zones.
Why can we not sequence thousands of DNA bases on a polyacrylamide gel?
Authors: Slater Gary W. , Drouin Guy 
Published: May 2005

AbstractPulsed fields have been remarkably useful at extending the range of DNA molecular sizes that can be separated on agarose gels by controlling the field‐induced molecular orientation that often limits the resolution of large molecules. Unfortunately, the same approach seems to be much less effective for DNA sequencing on polyacrylamide gels. We present an experimental and theoretical (modelling) study of DNA sequencing which shows that molecular orientation is indeed not the main limiting factor for sequencing devices that use moderate field intensities and polyacrylamide as a separating matrix. We examine the interplay between electric field intensity, molecular size and resolution, and we suggest different approaches to increase the resolution limit of standard and automated sequencing gels. The theoretical limits of high‐field electrophoretic sequencing are also discussed. We conclude that new ideas will be needed to go beyond one kilobase.
Localization of separated protein bands in unstained electrophoresed polyacrylamide gradient slab gel
Authors: Arora D. Jit S. 
Published: May 2005

AbstractPolyacrylamide gel electrophoresis has become the most widely used separation method in biological science. Once electrophoresis is complete the protein bands must be localized prior to excision. A zig‐zag gel cutter is described which cuts a strip of gel from the side of a slab gradient gel or a gel of uniform concentration in peaks and valleys. The location of the protein of interest is determined by counting the number of peaks on the stained side strip. The portion of the unstained gel corresponding to the same count (number of valleys) is cut to recover the protein of interest from the main gel for further manipulations.
Apolipoprotein E phenotyping by isoelectric focusing in immobilized pH gradients and silver staining
Authors: Cartier Régine , Sassolas Agnès 
Published: May 2005

AbstractA simple and high resolution procedure of apoprotein E (apo E) phenotyping by isoelectric focusing with immobilized pH gradients and silver staining is described. This method needs delipidated very low density lipoproteins (isolated from 1 mL of serum) but obviates immunoblotting as well as neuraminidase treatment in routine applications because the sialylated forms are clearly separated. Immunoblotting (with polyclonal and monoclonal anti‐apo E antiserum), cysteamine and neuraminidase treatment, and pI markers allowed the localization of three main alleles, ξ2, ξ3, ξ4 and the detection of variants or rare alleles (6/450 determinations). The serum amyloid A (SAA) apolipoproteins (SAA1, SAA2) could be characterized unequivocally (especially with E3 and E4). Silver staining proved more sensitive than Coomassie Brilliant Blue and needs only 5 μg of protein in the sample. The results of 403 normo‐or hyperlipidemic patients are shown. In the group of 191 normolipidemic patients (cholesterol < 6.40 mmol/L triglycerides < 2 mmol/L), the relative frequency of the ξ3 allele (0.83) is higher than in other reports on Caucasians (about 0.77) whereas the ξ4 allele is lower. As previously described, we find a high frequency of the 4/3 phenotype in hypercholesterolemia and 3/2 in hypertriglyceridemia. The high frequency of the E2/E2 phenotype, usually associated with hyperlipidemia, and variants in complex hypertriglyceridemia makes the apo E phenotyping necessary in many cases of dyslipidemias.
Calculation of restriction fragment lengths by image processing
Authors: Holmlund Gunilla , Karlberg Knut , Gustavsson Bengt , Lindblom Bertil 
Published: May 2005

AbstractThe image processing system, LabEye Profile, calculates the size of DNA restriction fragments according to the formula MD = f(x); x = 1/log bp. The migration distances (MD) of known fragment sizes are interpolated by the use of cubic splines. Cubic splines are also used to correct for gel distortions. A comparison to the Biotrac system, which uses the reciprocal method, gives almost identical standard deviations for calculated sizes compared to known sizes, 0.88% for LabEye Profile and 0.89% for Biotrac. The measurement error for LabEye Profile is 0.24% of the size of the fragments.
Elimination of sample diffusion and lateral band spreading in isoelectric focusing employing ready‐made immobilized pH gradient gels
Authors: Loessner Martin J. , Scherer Siegfried 
Published: May 2005

AbstractA simple procedure for the elimination of lateral sample as well as band spreading in precast, ready‐made immobilized pH gradient gels is described. Round or rectangular holes are punched in the dry polyacrylamide gels prior to rehydration. The generated wells proved suitable for application of samples containing surfactants such as Nonidet P‐40 or 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propane‐sulfonate (CHAPS). Lateral band spreading and precipitation of samples containing up to 9.5 M urea could be completely eliminated by this method.
Studies of DNA bending and flexibility using gel electrophoresis
Authors: Harrington Rodney E. 
Published: May 2005

AbstractGel electrophoretic methods have become established as primary tools in the study and elucidation of sequence‐directed curvature both in free DNA and in the operator DNA of several site‐specific nucleoprotein complexes. Results using them have been generally consistent with physical methods sensitive to DNA structure and conformation in those instances where direct comparisons can be made, and in a number of cases, gel methods have provided unique information not presently available from other techniques. Two basic strategies have been used: one based upon anomalous gel mobility effects; and a second based upon cyclization properties of curved DNA. Within each of these categories, various approaches have been used, some of which can lead, in favorable cases, to quantitative estimation of bending angles. In this review, the various gel‐based methods that have been used to date are critically discussed and the qualitative and quantitative information that can be obtained from them is evaluated. A number of possible structural models for DNA curvature are described and a distinction is drawn between static or fixed bending and bending due to anisotropic flexibility at specific sequence loci. The importance and roles of gel electrophoretic methods in providing experimental approaches to this question are discussed. It is suggested that both static curvature and anisotropic flexibility in operator DNA may provide much of the basis for indirect readout of sequence information by specific sitebinding regulatory proteins.
Capillary zone electrophoresis of p‐aminobenzoic acid derivatives of aldoses, ketoses and uronic acids
Authors: Grill Eduard , Huber Christian , Oefner Peter , Vorndran Andreas , Bonn Günther 
Published: May 2005

AbstractAldoses, ketoses and uronic acids were derivatized with p‐aminobenzoic acid and separated as their borate complexes by capillary zone electrophoresis, using a capillary tube of fused silica containing 150 mM borate buffer, pH 10.0, as carrier. The electrophoretic mobilities of 22 carbohydrates were determined and found to increase with increasing stability of the borate complexes formed. Besides the number of hydroxyl groups and the presence of substituents, complex stability depended most strongly on the configuration of the three vicinal hydroxyl groups at C2, C3 and C4. On‐column UV monitoring at 285 nm allowed the detection of glucose with a lower mass detection limit of 15 fmol and a concentration sensitivity of 4 μM. Reproducible quantification of carbohydrates was achieved at least in the concentration range of 0.1–10 mM in reaction solutions by the relative peak area method, using cinnamic acid as internal standard. The method was applied successfully to the determination of the monosaccharide composition of polysaccharides extracted from Radix althaeae.
Numerical simulation of protein separation by continuous‐flow electrophoresis
Authors: Clifton Michael J. 
Published: May 2005

AbstractContinuous‐flow electrophoresis is a process for separating protein mixtures on a preparative scale. Its resolution is determined by the migration distance at the collection plane and by the fineness of the filament occupied by each protein species. Filaments undergo spreading due to a number of different phenomena, among which electrokinetics and electrohydrodynamics are known to be important. In the first of these, differences in migration velocity between the ionic species give rise to local variations in pH and electrical conductivity near the protein filament. In the second, the local change in electrical conductivity distorts the electric field, thus inducing shear stress in the liquid and creating a local flow pattern. A numerical model has been developed to describe these phenomena when two proteins are being separated.
Two‐dimensional separations of peptides and proteins by comprehensive liquid chromatography‐capillary electrophoresis
Authors: Larmann John P. , Lemmo Anthony V. , Moore Alvin W. , Jorgenson James W. 
Published: May 2005

AbstractAnalysis of biological samples is problematic because of their complex composition. Reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), and, more recently, capillary zone electrophoresis (CZE) are routinely used for the analysis of these samples, but are eventually limited because they are one‐dimensional (1‐D) methods. As sample complexity increases, the separation efficiency necessary to resolve a large number of sample components in one dimension becomes prohibitively high. A solution to this problem has been to use a two‐dimensional (2‐D) approach. Each dimension in a 2‐D separation relies on a different separating mechanism. By expanding the separation into two dimensions, sample components unresolved in the first dimension can often be separated in the second. This circumvents the requirement for extremely high efficiencies in either dimension. Two‐dimensional slab gel electrophoresis has been used successfully in this area, but a more instrumental approach is desired. In this paper we describe three coupled‐column approaches to 2‐D separations. First, microcolumn SEC‐CZE is explored as a means of 2‐D protein analysis. Next, RPLC‐CZE is investigated for analysis of peptides in tryptic maps. Finally, RPLC is coupled with fast CZE (FCZE), a unique form of CZE analysis, for fast 2‐D analysis of peptides. Details of the instrumentation used in these 2‐D systems will be presented along with the results of some typical 2‐D analyses.
Application of high‐performance capillary electrophoresis to the analysis of H1 histones
Authors: Lindner Herbert , Wurm Martin , Dirschlmayer Arnold , Sarg Bettina , Helliger Wilfried 
Published: May 2005

AbstractHigh‐performance capillary electrophoresis for the separation of rat testis H1 histone variants and their phosphorylated modifications is described. The influence of buffer pH, hydroxypropylmethyl cellulose, and buffer concentration has been investigated. Under optimized conditions (500 mM phosphate buffer, pH 2, 0.03% hydroxypropylmethyl cellulose) using an uncoated capillary, eight H1 histone subfractions, including two H1° histones and H1t and their phosphorylated modifications, are resolved. Application of capillary electrophoresis to the separation of H1 histones provides an important new alternative to high‐performance liquid chromatography (HPLC) and traditional gel electrophoresis.
Potential and limitations of an optically active crown ether for chiral separation in capillary zone electrophoresis
Authors: Kuhn Reinhard , Wagner Joseph , Walbroehl Yvonne , Bereuter Thomas 
Published: May 2005

AbstractCapillary zone electrophoresis using optically active 18‐crown‐6 tetracarboxylic acid (18C6H4) as chiral selector was studied for the enantiomeric separation of primary amines. From the separation of a variety of pharmaceutical drug substances, amino alcohols and amino acids, conclusions could be made concerning the influence of the chemical structure of the analytes on the separation. In addition, the effects of experimental parameters such as pH, proportion of organic modifier and buffer composition on the separation are discussed. A synergistic effect obtained by the joint application of 18C6H4 and a cyclodextrin was exploited to resolve analytes which were separated neither by the crown ether nor by the cyclodextrin.
Transverse pore gradient gel electrophoresis, using the PhastSystem
Authors: Buzás Zsuzsanna , Wheeler David L. , Garner Mark M. , Tietz Dietmar , Chrambach Andreas 
Published: May 2005

AbstractThe application of pore gradient gels prefabricated for the PhastSystem (Pharmacia) to transverse pore gradient gel electrophoresis is demonstrated. It has the twofold advantage of (i) horizontal positioning, avoiding gel stretching during the preparation of these gels and resulting pore size irreproducibility experienced with vertically applied pore gradient gels, which necessitate an orthogonal transfer of spacers, and (ii) miniaturized gel dimensions, which allow a small sample load and a short duration of electrophoresis and staining.
The determination of cocaine and related substances by micellar electrokinetic capillary chromatography
Authors: Trenerry V. Craige , Robertson James , Wells Robert J. 
Published: May 2005

AbstractA quantitative method for the determination of cocaine and related substances by micellar electrokinetic capillary chromatography (MECC) is described. Quantitative results obtained for both monitor materials and a number of actual drug seizures by this new capillary electrophoretic method are comparable to a gas chromatography (GC) run in parallel, both in the values and coefficient of variation achieved. The advantages of this new method include minimal use of solvents, the ability to readily automate the procedure and the ability to quantitate illicit heroin seizures under the same conditions with the exception of detector wavelength alteration. The method has proved rugged and reliable for both heroin and cocaine in a number of inter‐laboratory proficiency studies.
Immobilized pH gradients: New pK values of acrylamido buffers in poly(N‐acryloylaminoethoxyethanol) matrices
Authors: Bossi Alessandra , Righetti Pier Giorgio , Chiari Marcella 
Published: May 2005

AbstractA novel matrix consisting of N‐acryloylaminoethoxyethanol, a hydrophilic monomer extremely resistant to hydrolysis, was recently reported by Chiari et al. (Electrophoresis, 1994, 15, 177–186). When using it as a matrix for grafting immobilized pH gradients for isoelectric focusing, a shift in protein spot position was noticed. This was attributed to a shift in pK values of the Immobiline buffers when changing from the standard poly(acrylamide) to a poly(N‐acryloylaminoethoxyethanol) matrix. A series of 1 pH unit gradients was constructed, where a single buffering Immobiline was used and titrated with a counterion having a pK removed by at least 3 pH units from the nearest extreme of the generated pH interval. It was noted that all compounds became weaker acids and bases, with a ΔpK ranging from −0.02 to −0.06 for the acids (pK 3.6, 4.4, and 4.6) and a ΔpK ranging from −0.12 tp −0.20 for the bases. The new pK values for the seven commercially available buffers are thus pK 3.6 ‐> pK 3.58, pK 4.4 ‐> pK 4.36, pK 4.51 ‐> 4.45, pK 6.21 ‐> 6.09, pK 7.06 ‐> 6.94, pK 8.50 ‐> pK 8.37, and pK 9.59 ‐> pK 9.39. These values refer to 10°C in the gel phase, the first value in poly(acrylamide) and the second in poly(N‐acryloylaminoethoxyethanol).
Quantitative studies of different injection systems in capillary electrophoresis
Authors: Ermakov Sergey V. , Zhukov Michael Yu , Capelli Laura , Righetti Pier Giorgio 
Published: May 2005

AbstractFor a rigorous assessment of the precise amount of sample loaded, for quantitation purposes, different sample injection systems were evaluated with two commercially available units, Waters Quanta 4000 and Beckman P/ACE 2100. In the first system, sample introduction by hydrostatic means (i.e., placing the sample vial at some height, usually 10.1 cm, above the other capillary end) was evaluated. It was found that in this system there is a constant positive bias, i.e. the amount of sample loaded lies on a curve parallel and above the theoretically predicted loading curve. However, the excess of mass loaded was constant along the injection times explored (covering from 5 to 35 s) and, for a 75 μm capillary, was found to be of the order of +6 nL (above the expected injected value). Thus it is easy to correct for this sample bias. In the electrokinetic mode, a very good correlation between expected and predicted sample loads was obtained for both units. In the pressure system (by positive pressure from a nitrogen tank, Beckman unit) a substantial discrepancy was found between experimental and predicted values (13.5% overload). Since the manufacturer claims a constant pressure of 0.5 psi, i.e. 3447.5 Pa, it would appear that the injection pressure is higher than the given value. Other causes for variation in sample load (e.g., as caused by diffusion of sample in the capillary just prior to injection, by the sudden insertion of the capillary tip into the sample vial, etc., lumped together in a general term as “extraneous injection”) have been evaluated and found to contribute to an additional sample volume injection of the order of 1–2 nL, i.e., quite negligible. In conclusion, it is felt that both the hydrostatic and electrokinetic injection modes are highly reliable. The positive pressure mode can fail due to leakage of O‐rings, valve malfunctioning and to the typical problems of mechanical systems. Thus, the latter injection system should be checked frequently for potential mechanical failures.
Separation of lipophilic compounds by micellar electrokinetic chromatography with organic modifiers
Authors: Otsuka Koji , Higashimori Mitsuo , Koike Ryo , Karuhaka Kaoru , Okada Yukihiro , Terabe Shigeru 
Published: May 2005

AbstractSeparation of lipophilic compounds such as polyaromatic hydrocarbons (PAH) by micellar electrokinetic chromatography (MEKC) with organic modifiers was investigated. Dimethyl sulfoxide (DMSO) and acetone were used as organic modifiers, and sodium dodecyl sulfate (SDS) as a surfactant or a micelle forming reagent. By using 25 mM SDS (pH 7.0), containing 50% v/v DMSO, 8 PAHs were separated. Similarly, with 25 mM SDS, containing 30% acetone, 13 PAHs were successfully separated. For the calculation of thermodynamic quantities, critical micelle concentrations of SDS in buffers containing DMSO or acetone were measured.
Spatial and temporal depletion of ions from noncrosslinked denaturing polyacrylamide in capillary electrophoresis
Authors: Figeys Daniel , Renborg Annika , Dovichi Norman J. 
Published: May 2005

AbstractElectrical conductivity across a polyacrylamide‐filled capillary decreases during the separation of DNA sequencing fragments. This conductivity decrease is localized to the first few centimeters at the injection (negative) end of the capillary; no conductivity change is noted at the detection (positive) end of the capillary. The zone of decreased conductivity extends further into the capillary as the separation proceeds. The zone is most important for freshly prepared capillaries; capillaries used nine days after polymerization generate an insignificant current drop. The data are consistent with ionic depletion due to differences in transport numbers between the separation medium and the buffer reservoirs.
Hybridization of the variable number of tandem repeat core oligonucleotide GNNGTGGG to 350 cosmids from chromosome 7
Authors: Melmer Georg 
Published: May 2005

AbstractAn efficient method using short oligonucleotides is described for the isolation of minisatellite markers of the type variable number of tandem repeats (VNTR). It is used to screen 350 cosmids from chromosome 7 for the presence of such potentially polymorphic DNA segments. From the number of hybridization signals to chromosome 7 cosmids, I estimate the human genome to have at least about 15000 VNTR loci containing minisatellite core sequences, which is well above previous estimates.