bluuebunny/crossref_metadata_2025_split huggingface dataset:
Silver staining of human body fluid proteins following two‐dimensional electrophoresis in the absence of protein denaturants
Authors: Marshall Thomas 
Published: May 2005

AbstractThe native protein patterns of human serum and unconcentrated cerebrospinal fluid, amniotic fluid, urine, sweat and tears have been detected by silver staining following two‐dimensional electrophoresis in the absence of protein denaturants. Over 100 proteins were detected in both urine (80 μl) and sweat (40 μl), thereby demonstrating the high sensitivity of the approach. However, optimal sensitivity was only achieved following pre‐equilibration of the gels in sodium dodecyl sulphate. The implications and possible nature of stain enhancement are discussed.
A new system for unloading gel rods for isoelectric focusing and a tank for running twenty second dimensional slab gels vertically
Authors: Ramasamy Ragini , Spragg Peter S. , Jones Miren I. , Amess Robert 
Published: May 2005

AbstractA stand is described which supports racks of gels for isoelectric focusing (ISO‐gels) and makes it possible to load these gel rods directly onto the second dimensional slabs of polyacrylamide (DALT‐gels). Equilibration with second dimensional buffer is made by adding it without agarose to the ISO‐gels on top of the slabs. A new design of DALT‐tank is also described to run twenty slabs of polyacrylamide while holding the plates vertically so the samples run down the slab. This system removes the need to seal the ISO‐gels onto the slab of polyacrylamide using agarose solutions. The omission of agarose reduces the background when two‐dimensional gels are stained with silver.
Unconcentrated human urinary proteins analysed by high resolution two‐dimensional electrophoresis with narrow pH gradients: Preliminary findings after occupational exposure to cadmium
Authors: Marshall Thomas , Williams Katherine M. , Vesterberg Olof 
Published: May 2005

AbstractOver 600 polypeptides were detected in the two‐dimensional patterns of unconcentrated human urine following ultrasensitive silver staining. Resolution was greatly improved using different carrier ampholyte (Ampholine) mixtures of narrow pH range during isoelectric focusing. The effects of freezing and thawing, dialysis, concentration and different protein denaturing procedures were investigated. Preliminary studies of the urinary protein patterns of workers occupationally exposed to cadmium for many years showed a pronounced but selective increase in the number and amounts of polypeptides of Mr <40 000.
Heating‐cooling plate for flawless open casting of agarose gels
Authors: Baumstark John S. 
Published: May 2005

AbstractA heating‐cooling plate for open casting of agarose gels is described. Use of this plate provides a flawless and convenient means of producing 0.8 mm or thicker gels of any size up to 25 × 30 cm on glass plates (0.5 mm thick gels tend to pull slightly away from the edge of the glass plate during pouring). Gels of of any thickness (0.5 mm or greater) can be prepared if GelBond film is used.
Glycosylation of human albumin in diabetes mellitus II. Extensive in vitro modification by trioses and hexoses as revealed by isoelectric focusing
Authors: Candiano Giovanni , Ghiggeri Gian Marco , Delfino Gerolamo , Queirolo Carlo , Gianazza Elisabetta , Righetti Pier Giorgio 
Published: May 2005

AbstractPurified human serum albumin (HSA) from normal adults has been reacted in vitro with different trioses and hexoses and the reaction products analysed by ultrathinlayer isoelectric focusing (IEF) under native and denaturing (8 M urea) conditions. The order of reactivity of these molecules has been found to be: glyceraldehyde > glyceraldehyde 3‐phosphate > glucosamine ≫ glucose ≫ acetaldehyde. By chemical analysis, the number of residues of glyceraldehyde bound to albumin appears high: 130 moles/mole of protein, while the number of exposed lysines is halved: ca. 8 in reacted vs. 16 in control albumin. It appears that a minimum length for an aldehyde to react with protein amino groups is three carbon atoms. A completely different IEF behavior has been found between “reacted and NaCNBH4 reduced” vs. “reacted and rearranged” HSA molecules. The former group, even when extensively reacted with sugars, exhibits a normal surface charge at pH = pI, possibly because, at the acidic pIs of HSAs, the now secondary (sugar‐bound) amino groups are still fully protonated. In the latter group, the bound sugar spontaneously undergoes an Amadori rearrangement followed by secondary reactions, still not fully clarified, which could possibly lead to ring structures (e. g. pyrrolic) with complete loss of positive charge from the sugar‐reacted amino groups. These species, in fact, show a markedly more acidic pI.
Capillary isotachophoresis of some isoquinoline alkaloids
Authors: Walterová Daniela , Stránský Zdeně;K , Preininger Vladimír , Šimánek Vilím 
Published: May 2005

AbstractThe isotachophoretic behavior of quaternary benzo/c/phenanthridine, protoberberine and aporphine alkaloids in different electrolyte systems is described. The concentration of the leading ion and the pH value of the leading electrolyte affect the relative effective mobilities of the alkaloids. The system of pH 4.7, containing the leading ion K+ (0.005 M) counter ion acetate, and the terminating ion β‐alanine (0.02 M), has been selected for the quantitative determination of the studied alkaloids in model mixtures and plant extracts.
Simple drying of polyacrylamide gels for fluorography and storage
Authors: Westermann Reiner 
Published: May 2005

AbstractA simple method for drying polyacrylamide gels of various size (0.3 mm capillary gels to 3 mm thick slab gels) and composition (T = 5–20 %, C = 0.7–5.3 %, sodium dodecyl sulfate gels, acetic acid/urea gels, isoelectric focusing gels) is described. The gels are destained in the presence of 20 % glycerol, dehydrated in acetone, and dried at room temperature. By this procedure, size reduced, clear or slightly opaque, and flexible sheets are obtained. The dried gels can be used for fluorography, stored for long periods and rehydrated without any cracking, warping, or loss in resolution. The shrinkage of the gels to about 25 % of their original size during acetone‐dehydration results in an increased staining intensity and shorter exposure times for fluorography.
Determination of molecular weights and Stokes' radii of non‐denatured proteins by polyacrylamide gradient gel electrophoresis 3. Estimation of the upper and lower size limits of carbonic anhydrase as a model for complexing enzymes
Authors: Purkhanbaba Huschang , Maurer Werner D. , Rothe Gunter M. 
Published: May 2005

AbstractStudying the separation behavior of various native carbonic anhydrase isozymes from mammalian erythrocytes we found that the migration of these enzymes differs from that of the marker proteins commonly used in gradient gel electrophoresis. In alkaline buffer systems the enzymes from human, bovine, rabbit, and canine erythrocytes start to migrate with a size apparently 6 to 12 times larger than their monomeric size, then gradually lose in apparent size and finally end up in a size equivalent to their monomeric mol mass. We determined the monomeric mol mass of the various carbonic anhydrase forms to be 23 000 to 39 000 (g/mol). These values are in accordance with different data in the literature and the data which we obtained by sodium dodecyl sulfate‐gradient gel electrophoresis. The equations and the graphical solutions needed to calculate the apparent upper size as well as the monomeric size of mammalian carbonic anhydrases are presented. It is demonstrated by this example that only the time‐dependent version of non‐denaturing polyacrylamide gradient gel electrophoresis provides reliable mol mass estimations.
Alterations in synthesis of the acid soluble proteins from nuclei of specific phases of the in vivo liver cell cycle after isoproterenol and sodium phenobarbital administration: An electrophoretic study
Authors: Pipkin James L. , Anson Jeanne F. , Hinson William G. , Robert Burns E. , Wolff George L. 
Published: May 2005

AbstractPartially hepatectomized mice were injected intraperitoneally with isoproterenol (IPR), sodium phenobarbital (PB), or a combination of the two, and the regenerating lobe of the liver was removed. Nuclei were isolated, electrostatically sorted from various phases of the cell cycle, and the acid‐soluble proteins were extracted. Synthesis of several high‐molecular weight non‐histone proteins were observed in two‐dimensional polyacrylamide gel electrophoretic autoradiographs after independent administration of IPR and PB. The histones revealed alterations in synthesis patterns and the histone variants demonstrated concentration changes in response to these drugs. A protein designated as “a” appeared in G0 + G1 phase nuclei after IPR treatment and showed an increase in concentration after PB was included in the dosing regime. This protein displayed homology with a non‐histone protein from calf thymus when its mobility and amino acid content was examined.
An improved protocol for two‐dimensional maps of serum proteins with immobilized pH gradients in the first dimension
Authors: Gianazza Elisabetta , Astrua‐Testori Silvia , Giacon Patrizia , Righetti Pier Giorgio 
Published: May 2005

AbstractAdditional information is provided on the performance of two‐dimensional electrophoretic separations of human serum using immobilized pH gradients in the first dimension first described in Electrophoresis 1984. 5, 209–216. Special attention is devoted to the effect of different sample manipulations, including the site of sample loading along the pH gradient, its concentration (moderate dilution has favorable effects for the quantitative and prompt migration of the proteins from the application well) and its pretreatment (different serum patterns result from incubation in various denaturing and non‐denaturing media). An interference to the migration from the first to the second dimension matrix is observed by high Immobiline concentrations (β power should then not exceed 3 mEq/liter pH). The presence of 2‐mercaptoethanol in the equilibration medium prior to the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis step is found essential for a proper peptide mapping.
Factors affecting the range of pH gradients in the isoelectric focusing dimennsion of two‐dimensional gel electrophoresis: The effects of reservoir electrolytes and loading procedures
Authors: Chambers John A. A. , Innocenti Francesco Degli , Hinkelammert Katharina , Russo Vincenzo E. A. 
Published: May 2005

AbstractWe have examined methods for broadening and stabilising pH gradients used in the first dimension of two‐dimensional gel electrophoresis. The replacement of typical strong electrolytes with weak electrolytes as reservoir anolyte and catholyte allows the generation of broad (3.5 pH unit) gradients that are stable for at least 28 000 volt × hours (Vh). Protein patterns form within 10 000 Vh are stable throughout the subsequent focusing. Large quantities of protein, up to 6 mg, can beloaded onto such gels by mixing samples into the gel prior to polymerisation. We found no evidence for extensive modification of proteins because of exposure to the polymerisation reaction. The pH gradient formed in strong electrolytes in the typical O'Farrell gel system (J. Biol. Chem. 1975, 250, 4007–4021) could also be broadened if the samples were again mixed into the gel before polymerisation. This last effect appears to be due to an effect of the sample buffer used upon end loading of samples.
Multiple crossed immunoelectrophoresis on agarosecoated GelBond film
Authors: Rochu Daniel , Fine Jean‐Marius 
Published: May 2005

AbstractA new technique, multiple crossed immunoelectrophoresis on agarose‐coated GelBond film, is described. A sheet of GelBond with agarose areas containing a single or different antisera can be used for the simultaneous analysis of eight samples. Antigens were separated in the first dimension (i) in an inert medium according to net charge (cellulose acetate membrane elecrophoresis), (ii) in a sieving medium according to charge and molecular size (ultrathin‐layer polyacrylamide gradient gel electrophoresis), or (iii) in a pH gradient according to differences in isoelectric points (ultrathinlayer isoelectric focusing). By using these methods in the first dimension, resolution is improved over that in conventional agarose electrophoresis. The first dimensional strips are easily transferred to the second dimensional antibody‐containing agarose. Use of the GelBond film allows easy handling and convenient storage. Multiple crossed immunoelectrophoresis is a versatile method for rapid analysis of antigenic variability.
Enhanced sensitivity of Gelcode silver staining by glutaraldehyde fixation of proteins in two‐dimensional electrophoresis
Authors: Slisz Mark L. , Van Frank Richard M. 
Published: May 2005

AbstractThe staining of proteins in polyacrylamide gels using Gelcode® silver staining can be enhanced approximately tenfold by fixing with glutaraldehyde. The glutaraldehyde results in the formation of a protein‐aldehyde complex. This complex retards the diffusion of low molecular weight proteins from the gel and increases staining sensitivity by increasing the amount of metallic silver deposited within the protein‐aldehyde complex.
Ionic mobility parameter
Authors: Michov Budin M. 
Published: May 2005

AbstractA linear equation representing the ionic mobility as a function of the ionic strength square root was worked out on the basis of our hypothesis for the existence of two ionic radii. The equation contains a proposed parameter, pi. Comparisons between this equation and the equations of Onsager, and Robinson‐Stokes were made, and the absolute mobilities of 20 ions at 0 and 25 °C were calculated according to it. It was demonstrated that equations derived from the proposed equation can be used for calculating electrolyte dissociation degrees and electrolyte concentration dissociation constants.
Antibody‐affinity blotting, a sensitive technique for the detection of α‐fetoprotein separated by lectin affinity electrophoresis in agarose gels
Authors: Taketa Kazuhisa , Ichikawa Eriko , Taga Hiroko , Hirai Hidematsu 
Published: May 2005

AbstractA sensitive technique of antibody‐affinity blotting was developed for the detection of small amounts of human α‐fetoprotein (AFP) in the presence of high background lectins and other serum proteins in agarose‐gel affinity electrophoresis. AFP was specifically transferred by blotting to nitrocellulose membranes which were precoated with affinity‐purified horse or goat polyclonal antibodies to human AFP. The AFP transfers were treated with rabbit immunoglobulins to human AFP, followed by affinity‐purified goat anti‐rabbit IgG(H+L)‐horseradish peroxidase conjugate for color development with 3,3′‐diaminobenzidine. The method allowed us to detect as low as 4 pg/mm2 AFP (or 4 ng/ml AFP with a 3 μl sample volume applied to a 0.5 × 6 × 1 mm trough). In lectin affinity electrophoresis, AFP bands constituting more than 10% of the total AFP were quantitatively detected with 5 μl of 125–500 ng/ml AFP applied to a 1 × 5 × 1 mm trough. When horse antiserum to AFP or its immunoglobulin fraction was used to precoat nitrocellulose membranes, the background stain due to concanavalin A(Con‐A) in agarose gel became marked. This was eliminated by washing the transfers with 0.2 M α‐methyl‐D‐mannoside before subsequent antibody treatments. The washing with α‐methyl‐D‐mannoside also counteracted the uneven staining of separated AFP bands by Con‐A. The antibody‐affinity blotting suits best for the quantitative transfer of a specific protein to nitrocellulose membrane in the presence of varying amounts of other background proteins.
FXIIIA Phenotyping by isoelectric focusing and immunoblotting: Gene frequencies in a population of US Whites and Blacks
Authors: Dykes Dale D. , Polesky Herbert F. 
Published: May 2005

AbstractPlasma specimens were phenotyped for FXIIIA in local Whites and Blacks using agarose isoelectric focusing in the range pH 4–7, followed by immunoblotting. Gene frequencies for the alleles FXIIIA*1, FXIIIA*2 and FXIIIA*4 in Whites were 0.774, 0.225 and 0.001, respectively. In Blacks FXIIIA*1 was observed at a frequency of 0.771 and FXIIIA*2 at 0.229. The rare phenotype FXIIIA 3‐1, which by conventional electrophoresis demonstrates two bands, was found to clearly show three bands using isoelectric focusing and immunoblotting. The technique used in this study is inexpensive, easy to perform and can differentiate the gene products of all the currently known variants of FXIIIA.
Factor B polymorphism: Analysis by isoelectric focusing in immobilized pH gradients followed by immunoblotting
Authors: Davrinche Christian , Charlionet Roland , Rivat Claude 
Published: May 2005

AbstractHuman factor B polymorphism has been investigated by isoelectric focusing in immobilized pH gradients in the range from pH 5.2 to 6.1, or 5.4 to 5.9, followed by nitrocellulose immunoblotting. Although the methodology offers a matchless mean to detect so far hidden factor B variability, we did not discover factor B subtypes while typing hundreds of fresh sera.
Characterisation of cultivars of Allium cepa L. (onions) by ultrathin‐layer isoelectric focusing of seed esterases
Authors: Cooke Robert J. 
Published: May 2005

AbstractA method for the extraction of multiple forms of esterase from onion (Allium cepa L.) seeds and their separation using ultrathin‐layer isoelectric focusing, is described. The reproducibility of the esterase patterns produced by the method is demonstrated. Potential uses of the technique for the characterisation of cultivars of onions are discussed.
Preparation of rehydratable polyacrylamide gels and their application in ultrathin‐layer isoelectric focusing
Authors: Frey Manuela D. , Kinzkofer Angelika , Bassim Atta M. , Radola Bertold J. 
Published: May 2005

AbstractA new approach to isoelectric focusing in polyacrylamide gels is described, based on the use of rehydratable gels which in dry form could be stored for extended periods and which prior to use were rehydrated with solutions of any composition. Ultrathin 60–240 μm polyacrylamide gels, with different composition (5 % T, 3 % C; 3 % T, 4 % C; 3 % T, 20 % C), were polymerized under well‐standardized conditions and, after polymerization, washed exhaustively with distilled water to remove any unreacted monomers, catalysts or soluble polymers. The washed gels were impregnated with suitable additives, before drying, to preserve gel functionality on storage. Polyol compounds, such as glycerol, sorbitol and dextran, as well as synthetic polymers like polyethylene glycol and polyvinylpyrrolidone, were the most efficient additives when incorporated into the gel in a concentration of 1–10 %, either as single substances or in different combinations. Prior to isoelectric focusing the dry gels were rehydrated to the original gel volume with a solution of carrier ampholytes, in some experiments with added separators or urea. Kinetic studies have shown rehydration, depending on gel thickness, to be complete within a few minutes. Isoelectric focusing in rehydratable gels was consistently more reproducible than in wet gels by such criteria as regularity of patterns and coalescence of marker proteins, including ferritin, applied at different positions. Rehydratable gels tolerated higher field strengths at the final stage of isoelectric focusing, with typical values of 500–900 V/cm and thus, at a given volt X hour product, equilibrium focusing could be attained in a shorter time, with improved resolution and sharper zones. Ultrathin‐layer isoelectric focusing in rehydratable gels proved insensitive to high salt concentrations (up to 0.5 M) of the samples. Rehydratable gels represent a new generation of gels, excelling over the traditional wet gels by better standardized properties, convenient handling and unsurpassed flexibility.
Quantitative densitometry of heat‐induced changes in whey proteins following ultrathin‐layer isoelectric focusing
Authors: Dannenberg Frank , Kessler Heinz G. 
Published: May 2005

AbstractThe effcient and rapid technique of ultrathin‐layer isoelectric focusing on 50 μm polyacrylamide gels was used for determination of heat‐induced changes of whey proteins (β‐lactoglobulin A and B as well as α‐lactalbumin) and a method for quantitative densitometry was developed. Improved reproducibility could be achieved by (i) introducing an internal standard (carbonic anhydrase), allowing for compensation for minor irregularities in gel composition, sample application or staining, (ii) running each sample in triplicate, and (iii) multiple scanning of each sample at different positions of the focusing track. Coefficients of variation of the integrated peak areas were approximately 3 % for β‐lactoglobulin A and B and about 6 % for α‐lactalbumin. Skim milk was heated in a pilot heating plant especially designed for reaction kinetic studies. Using the initial content of native whey proteins in each milk sample as a reference value, degrees of denaturation of more than 90 % could be determined for each of the three protein fractions. Results obtained from reaction kinetic studies revealed that denaturation of α‐lactalbumin was a first‐order reaction.
Identification of gamma‐glutamyltransferase in rat liver plasma membranes after two‐dimensional electrophoresis
Authors: Rahimi‐Pour Ali , Wellman‐Bednawska Maria , Galteau Marie‐Madeleine , Artur Yves , Siest GÉRard 
Published: May 2005

AbstractTwo‐dimensional electrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by silver staining or specific immunoblotting analysis, was applied to the characterization of gamma‐glutamyltransferase (GGT, EC 2.3.2.2.) in rat tissues. We confirmed that purified kidney GGT is a dimer composed of two non‐identical subunits with molecular masses of about 50 and 30 kDa. Both the light and the heavy subunits were separated into 6 and 2 protein bands, respectively. Antibody fractions against the 30 and 50 kDa subunits, purified by immunoaffinity from a whole antiserum directed to the rat kidney enzyme, recognized their corresponding subunits and did not cross‐react with each other. Studies of the reactivity of these antibodies towards GGTs from kidney and liver homogenates revealed an intraspecies dissimilarity in the molecular architecture of the kidney and liver GGTs, especially concerning the 30 kDa subunit. Following phenobarbital treatment of animals we observed an increase in immunoreactive GGT, accompanied by the appearance of additional polypeptides with more basic isoelectric points and higher molecular mass.
Computer analysis of two‐dimensional gels by a general image processing system
Authors: Tyson John J. , Haralick Robert H. 
Published: May 2005

AbstractIn order to survey changes in accumulation of several hundred proteins during the naturally synchronous nuclear division cycle of Physarum polycephalum, we have developed methods for analyzing two‐dimensional (2‐D) gel electrophoretograms using the general image processing system developed by the Spatial Data Analysis Laboratory at Virginia Tech. In this paper we describe fast and accurate methods for removing non‐homogeneous background intensity from a 2‐D gel image, for resolving overlapping protein spots, and for estimating the total integrated intensity in a protein spot by Gaussian modeling.