bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyacrylamide gel electrophoresis of small peptides
Authors: West Michael H. P. , Wu Roy S. , Bonner William M.
Published: May 2005

Two polyacrylamide gel electrophoretic systems for the separation of small peptides are described. In both systems the samples are concentrated by a buffer discontinuity, and the peptides separated according to size and charge in 40–50 % polyacrylamide resolving gels. The acidic gel has an operative pH between 3.0 and 3.1 while the alkaline gel has an operative pH between 9.1 and 9.2. The lower limit of resolution is about 100 daltons for a peptide with one positive charge in the acidic system. The radioactive peptides are detected by autoradiography. Peptides with a free amino group can be fixed in these gels with formaldehyde, so that fluorographic procedures can also be used for increased sensitivity. These techniques enable precise comparison of non‐derivatized peptides from complete protein fingerprints and offer a simple one‐dimensional analytical method for protein comparison, as well as a general method for separating and analyzing small peptides.
The electric field strength across isoelectric focusing gel slabs determined by an accurate and convenient method
Authors: Murel Andrei , Vilde Svetlana , Kongas Ago , Kirret Oskar
Published: May 2005

A simple and accurate method for the monitoring of the voltage distribution across horizontal slab gels in isoelectric focusing is described. Voltage is measured by passing a single Pt wire electrode (connected to an external voltmeter and to one of the electrodes through the power supply) through holes in the apparatus lid, bored at 5 mm distance from one another across the migration path. Conductances along the migration path calculated from direct voltage measurements are by one order of magnitude lower than those measured by conductimetry on gel slice eluates. Segmental resistances calculated from the measured voltages across adjacent holes in the lid during isoelectric focusing (IEF) add up to the total resistances across the gel obtained from the voltage and current readings on the power supply. In confirmation of previous reports, segmental resistance in IEF was shown to increase dramatically with time, with maxima shifting progressively from pH 4 to 5 in a representative pH3‐10 gradient. Joule heat production in IEF decreases with increasing molecular weight of the carrier ampholytes.
Electrophoretic mobility and isoelectric point of purified brush border membrane vesicles
Authors: Ohsawa Kazuaki , Ohshima Hiroyuki
Published: May 2005

The electrophoretic mobility of brush border membrane vesicles purified from the rabbit ileum by chromatography on porous glass beads has been measured with a Laser‐Zee microelectrophoresis instrument. The value obtained was measured in a solution containing 0.1 M NaCl, 10 mM HEPES‐Tris and 50 mM mannitol at pH 7.37 and 20 °C was ‐1.41 μm s−1 V−1 cm. The mobility depended on both pH and ionic strength of the solution. The isoelectric point was observed to be at pH 4.25. The brush border membrane vesicle surface was found to be highly negatively charged: there are 1000–2000 negative charges on each vesicle at ionic strength 0.1. Treatments with cation had an effect on mobility and gave a reversal of surface charge.
Electrophoretic characterization of human monocytes and lymphocytes before and after stimulation with concanavalin A
Authors: Bauer Johann , Hannig Kurt
Published: May 2005

Human mononuclear cells were split into platelets, monocytes and small lymphocytes by countercurrent centrifugal elutriation (CCE). Determination of the electrophoretic mobilities (EM) of each population revealed that total small lymphocytes, i. e. B, T and null cells, have an equal EM at 1.09 × 10−4 (cm2 V−1 s−1), whereas monocytes showed a symmetric distribution curve with a peak at 0.95 × 10−4 (cm2 V−1 s−1). Thus the cathodal shoulder of the EM distribution curve of total mononuclear cells is caused only by monocytes. Mononuclear cells were stimulated by concanavalin A. Growing and resting lymphocytes were isolated at different times by CCE and analysed by cell electrophoresis. In contrast to murine T cells, human Tlymphocytes did not change their EM within 3 days after mitogenic transformation.
The effect of phytohemagglutinin on the electrophoretic mobility of purple membrane particles
Authors: Kantcheva Maria R. , Popdimitrova Nezabravka G. , Stoylov Stoyl P. , Kovatchev Dimiter E.
Published: May 2005

Phytohemagglutinin (PHA)‐the lectin extracted from Phaseolus vulgaris‐is found to induce electrophoretic mobility changes of purple membrane particles. The effect is dose‐dependent. The ionic strength of the suspending medium modifies the reduction of the membrane surface charge density.
An improved electrophoretic transfer (electroblotting) apparatus
Authors: Shuttleworty Andrew D.
Published: May 2005

An improved apparatus for the electrophoretic transfer of proteins from gels to immobilising matrices is described. This device overcomes problems common to previous designs such as entrapment of gas bubbles within the gel sandwich during its assembly and from electrolytic gas generation during transfer. The apparatus has some novel features which facilitate its ease of use and enable several gels to be blotted at one time.
Determination of thiodiacetic acid in urine of people exposed to vinyl chloride by analytical capillary isotachophoresis
Authors: Křiváková Ludmila , Samcová Eva , Boček Petr
Published: May 2005

A method is introduced for the determination of thiodiacetic acid in urine by analytical capillary isotachophoresis with colunm coupling. Thiodiacetic acid is one of the final metabolites of carcinogenic vinyl chloride and appears at increased levels in the urine of people exposed to vinyl chloride vapours. The method enables the direct analysis of samples of 10 μl of urine without any pretreatment in about 45 min. The lowest detectable concentration of thiodiacetic acid was about 6 × 10−6 mol/liter and the reproducibility of the analyses in the range of 2 ‐ 15 × 10−5 mol. per liter was about 3 rel. %.
Sodium salicylate for fluorographical detection of immunoprecipitated proteins in agarose gels
Authors: Heegaard Niels H. H. , Hebsgaard Kim P. , Bjerrum Ole J.
Published: May 2005

Film detection of compounds labeled with the weak beta‐emitters 3H and 14C is demonstrated for electroimmunoprecipitated proteins in agarose gels by means of the water‐soluble fluorophor sodium salicylate. The degree of enhancement of film detection of tritium and carbon‐14 with 0.7 M sodium salicylate paralleled fluorography with diphenyloxazole on polyacrylamide gels. The method was optimized and the sensitivity, expressed as radioactivity per cm immunoprecipitate, was 0.9 nCi/cm (2000 dpm/cm) in 24 h for 3H and 0.4 nCi/cm (830 dpm/cm) in 18 h for 14C. The procedure was found superior to the commercially available enhancement solutions ‘EnHance’ and ‘Amplify’. Salicylate fluorography on Kodak X‐Omat L and AR‐5 films was about 30 and 60 times more sensitive than Ultrofilm 3H. With 14C‐labeled anti‐antibodies rocket electroimmunoprecipitation corresponding to about 50–100 pg protein could be visualized in 7 days. Conclusively, the method is rapid (soaking and drying require about 30 min), sensitive, reproducible, cheap, and advantageous if the immunoplates are to be stained for protein after fluorography.
The behavior of serum albumin upon isoelectric focusing on immobilized pH gradients
Authors: Gianazza Elisabetta , Frigerio Adele , Astrua‐Testori Silvia , Righetti Pier Giorgio
Published: May 2005

Isoelectric focusing on immobilized pH gradients dissociates most complexes between fatty acids and serum albumin from whole plasma. Most albumin thus migrates to a pI of 5.8, very close to the theoretical value of 6.0 as calculated with the Linderstrøm‐Lang equation. The phenomenon was elucidated through saturation experiments and time‐course monitoring of the protein pattern upon focusing.
Comparison of detection limits for various nitrocellulose binding immunoassays using β2‐microglobulin as a model antigen
Authors: Vissing Henrik , Madsen Ole D.
Published: May 2005

The detection limits for antigen in immunodot blots on nitrocellulose paper using β2‐microglobulin as a model antigen were determined. the binding of a mouse monoclonal antibody against β2‐microglobulin to various concetrations of purified human β2‐microglobulin was tested with six different methods. The most sensitive assay allowed the detection of about 40 pg/mm2 protein.
Silver staining of human body fluid proteins following two‐dimensional electrophoresis in the absence of protein denaturants
Authors: Marshall Thomas
Published: May 2005

The 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

A 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

Over 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

A 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

Purified 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

The 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

A 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

Studying 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

Partially 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

Additional 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

We 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

A 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

The 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

A 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

A 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

Plasma 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

Human 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

A 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

A 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

The 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

Two‐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

In 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.
Isoelectric focusing of dansylated amino acids in immobilized pH gradients
Authors: Bianchi‐Bosisio Adriana , Righetti Pier Giorgio , Egen Ned B. , Bier Milan
Published: May 2005

The 21 free amino acids commonly encountered in proteins have been transformed into “carrier ampholyte” species by reacting their primary amino groups with dansyl chloride. These derivatives can thus be focused in an immobilized pH gradient covering the pH 3.1 to 4.1 interval, except for arginine, which still retains a pI of 8.8. Due to their inherent fluorescence, the dansyl derivatives are revealed in UV light, with a sensitivity of the order of 2–4 ng/mm2. All nearest neighbors are separated except for the following couples: Asn‐Gln, Gly‐Thr, Val‐Ile and Cys‐Cys2, with a resolving power, in a ΔpI scale, of the order of 0.0018 pH units. Except for a few cases (notably the aromatic amino acids) the order of pI values is well correlated with the pK values of carboxyl groups, suggesting that the latter are not altered by dansylation. From the set of pKCOOH ‐pI values of the different amino acids, the pK of the tertiary amino group in the dansyl label has been calculated to be 5.11 ± 0.06. Knowing the pK of the amino‐dansyl and the pI of the excess, free dansyl label (pI = 3.34) we have derived, for its sulfonic acid group, a pK of 1.57.
Microelectrophoresis in the differential diagnosis of proteinuric diseases
Authors: Weber Michael H. , Cheong (Kim) Kyung‐Sun , Schott Klaus‐Joachim , Neuholff Volker
Published: May 2005

Micro‐polyacrylamide gel electrophoresis (micro‐PAGE) has been shown to be useful for molecular size separation of urinary proteins. In addition to the sodium dodecyl sulfate micro‐gradient gel electrophoresis in 10 μL capillaries, which is used routinely for examination of proteinuria in our laboratory, we tried micro‐gradient slab gel electrophoresis (micro‐slab‐PAGE) for molecular weight determination of urinary proteins. It was also employed for monitoring patients with proteinuric diseases. For detailed studied of urinary proteins, micro‐two‐dimensional‐electrophoresis is recommended.
High‐resolution two‐dimensional electrophoresis of myofibrillar proteins with immobilized pH gradients
Authors: Pernelle Jean‐Jacques , Chafey Philippe , Lognonne Jean‐Luc , Righetti Pier Giorgio , Bosisio Adriana Bianchi , Wahrmann Juan P.
Published: May 2005

Previous studies by chromatography and electrophoresis have demonstrated that myosin is composed of two classes of polypeptide chains: high relative molecular mass (Mr) components (200 000) and a class of lowerMrspecies, called the “light chains” (LC). It has been shown before by others that the slow‐twitching fibers contain 2 types of isoforms: LC1s and LC2s, while the fast‐twitching fibers contain 3 types: LC1f, LC3f and LC2f, the latter also existing as phosphorylated forms. In the present investigation, utilizing two‐dimensional (2‐D) maps generated with narrow immobilized pH gradients in the first dimension, a higher resolution of the myosin LC has been achieved. The LC1s chains are further fractionated into at least four isoforms; LC2s into three; LC2f into four and LC3f into three sub‐species in rabbit muscles. Skeletal muscle tropomyosin, which is separated into three forms (αs, αfand β), is here resolved into six components, while actin from skeletal muscle, previously described mainly as a single chain (α), is here sub‐fracionated into four isoforms of identicalMrand different charge. The series of isoforms here reported could represent a fine probe for investigating such complex phenomena as the cellular differentiation of human satellite cells from normal and pathological subjects in greater detail than before.
A novel highly sensitive electrophoretic method for the detection and quantification of proteinase activity
Authors: Poulsen Otto M. , Hau Jann
Published: May 2005

A novel highly sensitive method for the detection and quantification of proteinase activity is presented. The proteinases were forced electrophoretically into the substrate (sodium caseinate) containing agarose gel. The formation of enzyme‐substrate complexes retarded the migration of the proteinases in the electric field and decreased the overall mobility of the enzymes. The proteinases were released as a result of the degradation of the substrate, and consequently the height of the obtained rocket shaped zones of hydrolysis were proportional to the concentration of proteinases in the samples. The electrophoretic method accelerated penetration of the substrate‐containing gel by the proteinases and subsequently resulted in a 100‐fold increase in sensitivity as compared to the radial diffusion method, depending entirely on diffusion.
The voltage across wide pH range immobilized pH gradient gels and its modulation through the addition of carrier ampholytes
Authors: Fawcett John S. , Chrambach Andreas
Published: May 2005

In pH 4–10 immobilized pH gradient (IPG) electrophoresis, one observes (a) partial failure of the sample to enter the gel; (b) which halt protein migration; (c) absence of defined transient state band patterns; (d) lateral zone spreading and distorted zone profiles; (d) uneven voltage profiles with a distinct voltage maximum; (f) difficulty in measuring pH profiles with the contact electrode. These problems are remedied through the addition of 0.1 to 1 % carrier ampholytes (CA) to the IPG gel. Problems (a) to (c) disappear in IPG tube gels. IPG in presence of CAs (ICAPG) of pI‐range 3.5–9.5 or 6–8 yields a pH gradient, a protein pattern and a pH gradient stability essentially the same as that in IPG gels of the same pH‐range 4–10. ICAPG is insensitive to ionic strength of the sample up to 0.1 M. Gel swelling and water exudation increase in ICAPG which make ICAPG in tube gels inapplicable when CA concentrations exceed 0.1 %.
Size, charge and structural heterogeneity of Brucella abortus lipopolysaccharides demonstrated by two‐dimensional gel electrophoresis
Authors: Sowa Blair A. , Crawforda Richard P. , Heck Fred C. , Williams John D. , Wu Albert M. , Kelly Katherine A. , Garry Adams L.
Published: May 2005

Phenol extracted, alkali‐treated lipopolysaccharide (aLPS) from vaccine strain (S19) Brucella abortus was demonstrated by two‐dimensional gel electrophoresis to consist of at least ten silver staining, polydisperse analogues having different pIs. When tested on nitrocellulose immunoblots, all ten were antigenically reactive with bovine anti‐B. abortus polyclonal sera, but only six reacted with anti‐B. abortus O‐antigen murine monoclonal antibody. Analogues focusing at different pIs were concluded to arise from differences in either core or O‐antigen side chain structure or because of covalently bound protein. While not qualitatively different, aLPS from pathogenic B. abortus strain 2308 had lesser amounts of analogues 1, 2, 5, 6, and 8 than did aLPS from strain 19 (vaccine). The 2‐D gel electrophoresis method was demonstrated to be of value in the analysis of aLPS from B. abortus and may be useful in the study of lipopolysaccharides from other sources.
HERMeS: A second generation approach to the automatic analysis of two‐dimensional electrophoresis gels Part I: Data acquisition
Authors: Vincens Pierre , Paris Nicolas , Pujol Jean‐Luc , Gaboriaud Christine , Rabilloud Thierry , Pennetier Jean‐Louis , Matherat Philippe , Tarroux Philippe
Published: May 2005

In this series of papers, we describe the computer system we have developed for two‐dimensional (2‐D) gel analysis. Here the acquisition part of the system is described and a set of criteria able to measure the overall performance of the system is presented. These criteria are essentially based on frequency domain considerations using Fourier transforms. We show that the transfer function characteristics of the input system must be carefully checked to avoid any image distortion; we propose an easily computed criterion to obtain setting conditions giving a correct system response. These features are discussed considering the specificity of the 2‐D gel images that can be well modeled by a multi‐Gaussian surface. Then the visualization device and its main abilities to treat and modify the aspect of the images are presented. Statistical functions using second‐order image histograms are introduced. They can be used to look at the general aspect of the images, as a global and accurate criterion for quality checking. For this purpose, covariance, entropy and inertia have been found to be most suitable. Second‐order histograms give additional information on image anisotropy and can direct the user towards the convenient values of the parameters for the subsequent treatments. Coding is considered from both the point of view of image compression and image sending throughout a computer network. An efficient code protecting all the image informations is suggested and discussed.
Detection of soluble peroxidases by direct plant tissue isoelectric focusing
Authors: Vioque Blanca , López Antonio , Castellano José M. , Albi Miguel A. , Vioque Agustín
Published: May 2005

Rectangular sections of leaves, vacuum‐infiltrated with distilled water or aqueous solutions of potassium chloride, were applied directly onto the surface of polyacrylamide gels followed by isoelectric focusing. Soluble peroxidases (free and ionically bound) were released from the tissue on isoelectric focusing with resultant characteristic zymograms for the different plant tissues.
Reducing the background to silver‐stained polyacrylamide gels after two‐dimensional electrophoresis
Authors: Amess Robert , Peter Spragg S.
Published: May 2005

A high background found after silver staining of polyacrylamide gels following two‐dimensional electrophoresis and generated from the cathode in the second‐dimensional separation has been investigated. Material is formed which is electrophoretically mobile and apparently derived from the buffer components. It behaves like a peptide as far as silver staining. The intensity of this background was reduced by treatment of the second‐dimensional gel with a photographic fixing agent prior to silver staining. Moreover, spots which previously appeared negatively stained were changed by the treatment and the result was a general improvement in the contrast.
Apolipoprotein E phenotyping from serum by Western blotting
Authors: Menzel Hans‐Jürgen , Utermann Gerd
Published: May 2005

A new method for the apolipoprotein E (apo E) phenotyping directly from human serum has been developed. The method is based on isoelectric focusing of delipidated serum in vertical slab gel systems followed by blotting and immunostaining using polyclonal anti‐apo E antibodies as the first antibody. Apo E phenotyping with this method in 150 serum samples gave exactly the same results as obtained with the conventional method based on isoelectric focusing of delipidated very low density lipoproteins isolated by ultracentrifugation and detected by protein staining. Compared with the conventional method, the present method is less expensive, suitable for large‐scale screening purposes e. g. diagnosis of type III hyperlipidemia and needs only a few microliters of serum. By application for screening of hyperlipidemic sera, we were able to detect two patients with an apo E‐1 variant and two others with “new” E‐3 and E‐4 mutants by the described methods. In addition apparent molecular weight variants of apo E could be detected by sodium dodecyl sulfate‐gel electrophoresis of sera followed by blotting and immunostaining.
Combination of line immunoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for molecular weight evaluation of isozymes: Application to iso‐α‐amylases identified in barley and rice seeds
Authors: Kerhardy Frédéric , Hara‐Nishimura Ikuko , Nishimura Mikio , Daussant Jean
Published: May 2005

A procedure for enzyme purification by means of line immunoelectrophoresis compatible with subsequent sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified enzyme and Coomassie Blue staining is described. Electrophoresis was carried out in an agarose gel backed to GelBond film. The enzyme activity was detected after immunoelectrophoresis on the wet gel and the immunoprecipitate by protein staining on the dried gel. The strip bearing the maximum amount of precipitate was excised and the dried agarose gel scratched off the GelBond support. The dried gel was extracted with the Laemmli sample buffer, heated at 100 °C for 10 min and submitted to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Proteins were stained with Coomassie Blue. Difficulties due to the presence of the immunoglobulin G subunits were encountered. The validity of the procedure was checked with an α‐amylase from barley purified by immunoaffinity chromatography. The procedure was used to compare the molecular weight of α‐amylase isozymes identified in barley and in rice seeds. In barley, α‐amylase I, the minor enzyme group appearing during germination, had a slightly higher molecular weight than α‐amylase II, the main enzyme group. In germinating rice seeds, the minor enzyme group, antigen D, was found to have a molecular weight slightly lower than the one of the other rice α‐amylases. A new α‐amylase antigen identified in rice as the constituent with the lowest pI was called pre‐A. Its molecular weight was found to be the same as the one of the main enzyme group, antigen (A + B).
HERMeS: A second generation approach to the automatic analysis of two‐dimensional electrophoresis gels. Part IV: Data base organization and management
Authors: Vincens Pierre , Tarroux Philippe
Published: May 2005

A database structure is needed for easy handling of the large quantity of data obtained from a series of gel electrophoresis experiments and also for defining the first statements of a standard between experiments from different origins. This paper describes the database concept and the database interrogation strategies in the field of two‐dimensional electrophoresis and protein expression data. First, the main characteristics of our database structure and the main capabilities offered to the user to handle and interrogate the structure, as well as to retrieve his information are described. Gel standardization and protein identification are then examined. A relational database, associated with the description of each spot according to internal and external (physicochemical) descriptors, provides a suitable structure for a standardization procedure. A dynamic updating of the actual position of each spot, each time new positional information is entered in the system, allows the encoding of standardized positions. A database structure can also be used to establish a link between different kinds of information from different areas required for a correct interpretation of two‐dimensional gel data. Particularly, information from the gene sequence of a given protein may offer insights into the regulation of this protein. Thus a link between the two‐dimensional gel database and sequence databases is implemented in our system. The information available from sequence data may help to interpret electrophoresis data. Furthermore, a formal presentation of the database structure is achieved in order to propose a standardized description of the database structure and concepts used. This feature allows comparisons between different databases and frees the user from a specific configuration. Logical formulations are introduced using predicate calculus and then lead to a description of manipulation tools in terms of a declarative programming language.