bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of rat liver carboxylesterase isozymes (EC 3.1.1.1) using polyacrylamide gel electrophoresis and isoelectric focusing
Authors: Simon Babette , de Looze Stephen , Ronai Adam , von Deimling Otto
Published: May 2005

The banding pattern of rat liver carboxylesterases (EC 3.1.1.1) was demonstrated following polyacrylamide gel electrophoresis and isoelectric focusing using standardized conditions. Phenotypic variations, occurring in commonly used inbred rat strains, were compared. Separate isozymes were identified using genetic nomenclature. Individual bands were labelled; their electrophoretic parameters were estimated. Three hitherto genetically undefined zones were described and preliminarily classified as carboxylesterase isozymes. A scheme was provided to enable identification of liver esterases in rat strains not investigated in the present study.
Two‐dimensional gel analysis of urine proteins after acidified‐acetone extraction
Authors: Guevara Juan G. , Herbert Barbara H. , Martin Barbara A.
Published: May 2005

An effective, reproducible and simple method for separating proteins from other urine constituents is described. Approximately 95 % of detectable urine protein is collected in the void volume peak from a single molecular exclusion gel column following an acidified acetone extraction of lyophilized urine. Analysis of the final urine protein preparation, using two‐dimensional (2‐D) gel electrophoresis and staining with silver showed patterns comparable to those reported by others. An estimated 700 individual proteins were discernible in 2‐D electropherograms of 75 μg of urine proteins. This is a reliable procedure that makes possible the isolation of proteins excreted in urine, which can then be used in other types of analysis or in further purification of urinary antigens.
Suitability of sulfur labelling reagent (Amersham) as an in vitro protein radiolabelling reagent for two‐dimensional electrophoresis
Authors: Rabilloud Thierry , Therre HéLène
Published: May 2005

This report describes a method of in vitro labelling of proteins designed for subsequent analysis of the sample by two‐dimensional electrophoresis. As much as 106 cpm can be incorporated in 5 μg of proteins. As the reagent reacts with the amino groups, virtually all the proteins are labelled. Thus, this method is far more sensitive than silver staining and may prove useful when extremely low amounts of sample are to be analysed.
Computer simulation of immobilized pH gradients at acidic and alkaline extremes: A quest for extended pH intervals
Authors: Mosher Richard A. , Bier Milan , Righetti Pier Giorgio
Published: May 2005

Computer simulations of the concentration profiles of simple biprotic ampholytes with Δ pKs 1, 2 and 3, on immobilized pH gradients (IPG) at extreme pH values (pH 3–4 and pH 10–11) show markedly skewed steady‐state profiles with increasing kurtosis at higher Δ pK values. Across neutrality all the peaks are symmetric irrespective of their Δ pK values, but they show very high contribution to the conductivity of the background gel and significant alteration of the local buffering capacity. The problems of skewness, due to the exponential conductivity profiles at low and high pHs, and of gel burning due to a strong electroosmotic flow generated by the net charges in the gel matrix, also at low and high pHs, are solved by incorporating in the IPG gel a strong viscosity gradient. This is generated by a gradient of linear polyacrylamide which is trapped in the gel by the polymerization process.
Two‐dimensional gel electrophoresis of cerebrospinal fluid immunoglobulins
Authors: Wiederkehr Felix , Ogilvie Allan , Vonderschmitt Dieter J.
Published: May 2005

With the aid of two‐dimensional electrophoresis, the immunoglobulins (Ig) in cerebrospinal fluid (CSF) were separated into heavy and light chains and visualized with silver staining. CSF from 36 patients with no abnormal immunologic activity in the central nervous system show a constant pattern of light chain subfractions, however varying strongly in their respective quantities. In 40 patients with inflammatory diseases such as confirmed multiple sclerosis (MS) (n = 10), neurosyphilis (n = 6), lymphocytic meningoradiculitis (n = 3), novel immunoglobulins appear and form a so‐called oligoclonal zone in the light chain region. By comparing various oligoclonal zones, differences were found between the arrangement of the novel Ig light chains and the polyclonal light chain background. In the region of the polyclonal light chain background certain protein fractions appear which are non‐immunoglobulin and which are not found in serum. The protein fraction at the isoelectric point of 7.8 appears in 9 out of 10 confirmed MS cases, but in none of the 6 neurosyphilis and 3 lymphocytic miningoradicultis cases. In one case of neurosyphilis with an extremely high gamma‐globulin content in CSF we found an oligoclonal zone in the heavy chain region.
Ready identification of disulfide‐bonded polypeptides by one‐dimensional sodium dodecyl sulfate‐polyacrylamide slab gel electrophoresis
Authors: Gurusinghe Asitha , Ryan Patricia A. , Davis Paul F.
Published: May 2005

A method for the detection of disulfide bonded polypeptide components in protein mixtures is described. During the electrophoresis of proteins in sodium dodecyl sulfate‐polyacrylamide slab gels, the migration is interrupted and advantage is taken of the rapid diffusion of added 2‐mercaptoethanol into lanes without reductant. In this paper, the results of applying this procedure to proteins known to contain only inter‐chain (α2‐macroglobulin, fibrinogen, type III collagen) and intrachain disulfides (bovine serum albumin, alkaline phosphatase) as well as to crude extracts are reported.
Sample band spreading phenomena in ground and spacebased electrophoretic separators
Authors: Rhodes Perey H. , Snyder Robert S.
Published: May 2005

Sample stream spreading in the transverse direction (direction perpendicular to the chamber center plane) is calculated for two ground‐based and one space‐based continuous flow electrophoresis chambers. The magnitude of transverse sample stream spreading is shown to be a function of the difference in electrical conductivity between the sample and curtain buffer. Sample/buffer conductivity mismatch distorts the normally uniform electrical field in the vicinity of the sample to produce transverse field components which, in turn, deflect sample material toward the chamber walls. These results indicate that non‐gravity effects are important and must be resolved in order for space‐based continuous flow electrophoresis to achieve its maximum potential.
Mathematical approach to zone boundary of isotachophoresis system
Authors: Shimao Kazuo
Published: May 2005

A mathematical approach to the behavior of isotachophoretic and counter ion components in the steady state of isotachophoresis of weak acids was developed. In steady state, the partial time derivative can be replaced by the ‐ν times χ derivative, where ν is the velocity of isotachophoresis. Application of this relation to mass balance equations of the components gave the expression of concentration gradients as functions of concentrations of the components, conductance and the H+ ion concentration gradient. The results were inserted into the χ derivative of the electric neutrality equation and the electric current equation, including both electrophoretic and diffusional currents. These equations were solved for conductance and H+ ion concentration gradients and the latter quantities could be expressed as functions of concentrations of the components. Thus, concentration gradients of the components were calculable from the concentrations. Given proper initial or boundary conditions for the concentrations of the components, numerical integration gave pH, conductance and concentration profiles of the components. The results were in agreement with those of capillary isotachophoresis experiments.
Micro‐isoelectric focusing of single pollen grains from Cucurbita pepo L
Authors: Gay Gilles , Kerhoas Catherine , Dumas Christian
Published: May 2005

A technique of isoelectric focusing of proteins from single pollen grains has been developed using ultrathin polyacrylamide gels. On a single gel, one hundred grains may be compared for total proteins or enzyme patterns. With this technique it was possible to reveal heterogeneity of pollen grains in a population at a genetic level and in relation to pollen viability.
Specifying the equilibrium constants in Tris‐borate buffers
Authors: Michov Budin M.
Published: May 2005

The pK values of the chemical processes which take place in Tris‐borate buffers are determined with higher accuracy using more precise Tris ion and boric acid pK values, and the buffer capacities at different pH values are calculated. The results confirm that Tris‐borate buffers contain a complex compound with betainic structure previously described as “Tris‐boric” acid.
Diagonal isoelectric focusing on immobilized pH gradients: Concept and preliminary performance tests
Authors: Fawcett John S. , Chrambach Andreas
Published: May 2005

Electrofocusing on diagonal immobilized pH gradients was designed to combine high field strength with a flat pH gradient over a wide pH range, circumventing the need for extremely high voltage in long, conventionally oriented focusing gels. The technique was found to be applicable at 500 V cm−1 to gels of 35 cm length of a flat pH gradient with a predicted pH range of 3.8 to 9.8, allowing for a focusing time of 2 h. Note that the equivalent flat pH gradient gel of 25 cm length at the same field strength would require application of 12 500 V. However, preliminary results indicate that the theoretically expected improvement in resolution due to high field strength does not materialize. This may be due to a distorted electric field caused by non‐uniform conductance features inherent in the method and by the passage of the current through the isoelectric protein zones.
Application of an electrophoretic method for the detection of acid, neutral and alkaline proteinases following gel filtration of the crude proteinase system of Aspergillus oryzae
Authors: Poulsen Otto M. , Hau Jann
Published: May 2005

A novel electrophoretic method for detecting proteinase activity was used in order to study the column chromatographic separation of different acid, neutral and alkaline proteinases of a commercial crude proteinase preparation of Aspergillus oryzae (Rhozyme 41). Fractions obtained by gel filtration of Rhozyme 41 on Sephadex G‐100 were analyzed by the electrophoretic method and the radial diffusion method at pH 4.6 and pH 8.6. The electrophoretic method made it possible to discriminate between various acid, neutral and alkaline proteinases due to differences in the morphology of the clearing zones, whereas no differences in the morphology of the clearing zones could be observed when using the radial diffusion method.
Analysis of proteins in renal tubule fluid by ultramicrogradient gel electrophoresis in 0.5 microliter capillary tubes
Authors: Manz Nany , Galaske Rainer G. , Cho Joan , Van Liew Judith B.
Published: May 2005

A research method is described for producing ultramicrogradient acrylamide gels in 0.5 μL constant volume capillary tubes (inner diameter 130 μm and 32 mm length) suitable for the determination of protein in kidney tubule fluid. Previous methods have been limited to gels contained in 1 and 5 μL capillary tubes. The acrylamide gel concentration varies from 4 to 27% in a functional gel of approximately 40 nL and a length of 2.0 to 2.9 mm. Relative migration distances in these gels of known proteins of varying molecular weight (6000 to 669 000) are comparable to those measured in gels contained in 5.0 μL capillary tubes. After electrophoresis, the gels are extruded from the capillary tubes, stained with Fast Green, destained and scanned in a Joyce‐Loebl microdensitometer with integrator unit. Such gels are capable of retaining and quantitating low molecular weight proteins contained in nanoliter volumes of biological fluids. The reproducibility of this technique for 0.5 μL capillary gels is comparable to that achieved with the routine analysis of proteins in unconcentrated urine using 5.0 μL gels. Examples are given of protein analyses in kidney tubule fluids and urine obtained from normal rats and those with glomerulonephritis.
The development of an automated cell electrophoresis analyzer
Authors: Fujii Hidehiko , Arisawa Takeshi , Akiyama Jun'Ichi , Yamada Takashi
Published: May 2005

An automated cell‐electrophoresis apparatus is described which appears suitable for clinical laboratory applications. A solid state laser is employed; however, the instrument departs from the usual Doppler systems in that coherence is not required and stationary transmission grating optics are used. A dedicated microprocessor performs fast Fourier transforms (FFT), curve fitting for pedestal and low frequency (LF) noise removal, and digital filtering to suppress FFT artifacts. Unique features include fully automated sample handling, and measurements are made at the center of the migration tube in lieu of the “stationary layer” for enhanced signal quality. Experiments show high resolution of spectra from individual and mixed populations of sheep and rabbit erythrocytes. The sample‐to‐report cycle of 5 or 7.5 min (switchable), high cost/performance ratio and elimination of potential operator errors make the instrument a candidate for clinical use.
Thermodynamic analysis of the interactions of a mouse dinitrophenyl‐specific myeloma protein, MOPC 315, with immobilized dinitrophenyl and trinitrophenyl ligands by affinity electrophoresis
Authors: Tanaka Tatehiko , Suzuno Ryosuke , Nakamura Kazuyuki , Kuwahara Akira , Takeo Kazusuke
Published: May 2005

Affinity electrophoresis was applied to determine the dissociation constants and the thermodynamic parameters for the interactions between the mouse myeloma, MOPC 315, and trinitrophenyl (Tnp) and dinitrophenyl (Dnp) haptens. The myeloma protein consists of monomeric, dimeric, and trimeric forms of IgA. The dimer had a higher affinity to the haptens than the monomer, but its Fab' fragment had lower affinity than the monomer. All of these myeloma proteins had higher affinities to Tnp‐hapten than to Dnp‐hapten. The affinity of MOPC 315 proteins increased when the temperature increased. The van't Hoff plots for all of these interactions gave straight lines within the temperature range from 7 °C to 40 °C. The ΔH° were calculated by using the linear van't Hoff plots. The ΔS° were calculated from the D̊H° and D̊G°. The D̊H° and D̊S° were both positive. Hence, the interactions were endothermic and hydrophobic.
Detection of neutral amino acid mutations by immobilized pH gradients: The case of the Tγ variant in fetal hemoglobin Sardinia
Authors: Cossu Gianfranco , Manca Mario , Righetti Pier Giorgio , Gianazza Elisabetta , Baudin VÉRonique , Wajcman Henri , Bianchi‐Bosisio Adriana
Published: May 2005

A method for separating fetal hemoglobin Sardinia (AγT, a variant containing threonine in position 75 instead of isoleucine) from normal fetal hemoglobin is described, based on the use of shallow immobilized pH gradients (pH 7.30–7.55). The novelty of the fractionation technique is the direct analysis of intact, native hemoglobin tetramers, while present methods utilize only reverse‐phase, high performance liquid chromatography of denatured, heme‐free globin chains.
Reproducibility of immobilized pH gradients after seven months of storage
Authors: Altland Klaus , Becher Pia
Published: May 2005

Recently we described a procedure for pouring immobilized pH gradients at ambient temperature in the presence of a neutral buffer (0.1 M Tris‐phosphate pH 6.8) (Altland and Rossmann, Electrophoresis 1985, 6, 314–325). After washing in plain water and in 1 % glycerolin water, the gels were dried, sealed in polyethylene bags and stored at –20°C until use. The reproducibility of a batch of 20 gels with a linear pH 6–10 gradient, poured under computer control, was studied by rehydrating the dried gels in a solution containing 8 M urea, 50 mM dithiothreitol and 0.5 % carrier ampholytes (pH 6–10) and by running human red cell lysates as samples. A comparison of the globin patterns obtained at various time intervals within a range of 7 months revealed that there was no detectable shift in the pH gradient within this period and that gels stored for 7 months can be used to clearly identify all human globins from newborns and adults. Since the tested pH gradient was prepared from Immobilines claimed as being the most sensitive to degradation by hydrolysis (e. g. Immobiline pKa 8.5 and 9.3), it is concluded that storage of dry gels, rehydrated prior to use, is a realistic alternative to the preparation of fresh gels from stored stock solutions for achieving reproducible results in long‐term comparative studies.
Simplified procedure for the preparation of immobilized pH gradient gels
Authors: Fawcett John S. , Chrambach Andreas
Published: May 2005

Immobilized pH gradient (IPG) slab gels are conventionally formed by use of two‐chamber gradient mixers on pH‐neutralized Immobiline mixtures, co‐polymerization of the gradient of Immobilines with acrylamide at 50 °C, washing and drying of the gel to its original weight followed by pre‐electrophoresis before applying the sample. This tedious procedure was replaced by one using gradient formation by pump, eliminating pH neutralization of the monomer mixture, substituting polymerization at 50 °C for one at the temperature of electrophoresis (10 °C in this study) and omitting washing and drying of the gel prior to use. Carrier ampholyte (CA) containing IPG gels were formed in the same way except that CAs were added to the polymerization mixture. Pre‐electrophoresis of IPG gels, formed by either the conventional or the simplified procedure, was found to be detrimental to the IPG patterns of proteins. The simplified procedure also allowed one to conduct IPG electrophoresis in gel tubes, eliminating lateral zone diffusion.
The use of biotinylated DNA probes for detecting single copy human restriction fragment length polymorphisms separated by electrophoresis
Authors: Dykes Dale , Fondell Joseph , Watkins Paul , Polesky Herbert
Published: May 2005

Human genomic DNA probes labeled by nick translation with biotin 11‐UTP were successfully used to identify single copy restriction fragment length polymorphisms following electrophoresis and Southern blotting onto nylon membranes. Two human probes found on chromosome 21 were used, each of which demonstrated polymorphisms using two separate restriction endonucleases. Compared to isotopic labeling of probes the biotin labels are safer, faster and more economical. The detection method of streptavidin‐biotin‐alkaline phosphatase permitted phenotyping in a matter of hours after hybridization of the probe to the human DNA fragments.
A program in BASIC for the determination of molecular weights and linear graphic representation of an electrophoretic protein pattern: Application to serotypes A and B of Candida albicans studied by polyacrylamide gel electrophoresis and immunoblotting
Authors: Ibrahim‐Granet OumaïMa , Delga Jean M. , Granet Christian , Drouhet Edouard
Published: May 2005

A program in BASIC Microsoft for a microcomputer is described for the analysis of electrophoretic patterns obtained by polyacrylamide gel electrophoresis. The program enables a rapid calculation of relative molecular masses and designs three types of graphs: the protein pattern, the linear representation of molecular masses and the linear regression curve calculated from several standard proteins. The application of this program for the study of serotypes A and B of the yeast Candida albicans is shown.
Frequency effects in protoplast dielectrophoresis
Authors: Stoicheva Natalia , Dimitrov Dimiter S.
Published: May 2005

Pea protoplast dielectrophoretic coefficients were measured in AC fields of different frequencies applied between concentric cylindrical metal electrodes, outer and inner radii, 0.24 mm and 1 mm, respectively. The values for 0.5 M mannitol solutions are: (6.0, 1.0, 5.6, 16.0 and 17.8) X 10−24Fm2 at frequencies 0.05, 0.1, 0.6, 1 and 8 MHz, respectively. The specific polarizabilities (dielectrophoretic coefficients per unit volume of the protoplasts) are: (5.0, 0.7, 4.0, 12.0 and 13.0) X 10−10Fm−1, respectively. Below 10 kHz dielectrophoretic effects were not observed. Cell rotation was observed in the frequency range from 1 kHz to 10 kHz. These results may be of use in cell separation and electrofusion in scientific research and technology.
HERMeS: A second generation approach to the automatic analysis of two‐dimensional electrophoresis gels Part II: Spot detection and integration
Authors: Vincens Pierre
Published: May 2005

This work describes the techniques used to detect the presence of a spot at a given place in a two‐dimensional gel and to compute its volume. This is done using filtering techniques to extract significant information from rough images. Four types of treatments were applied to images in order to improve the detection of spots belonging to different categories. Mathematical techniques of morphology were used to lower the effect of spot overlapping, while histogram modifications using power functions were used to improve the detection of major and minor spots, respectively, thereby generating four processed images. On each generated image, spot detection is computed by looking at the local maxima. Then the four spots lists are gathered and the volume is computed according to a spot model. To improve the accuracy of volume detection, a spot model constituted by two half‐Gaussian curves in each space direction is used. The pitch of each spot according to the horizontal direction is also computed. A total of 8 parameters, including spot position, shape parameters, surface intensity and volume, is gathered into a spot list which constitutes the reduced information which will be handled subsequently. The choice of a model allows us to get a round the improper volume calculations caused by spot overlapping. The mutual penetration of one spot into another is thus automatically taken into account. A set of statistical considerations is computed from these data in order to validate our spot model, to check for parameter independence, to obtain quality criteria which may be used to judge gel quality, to propose different diagnoses for the observed defaults and to lead to a self‐adaption process of treatment parameters. These statistical considerations can be used to study the effect of each type of treatment and to compare the effects of each filter on the final result.
Immunoblotting analysis of the peptide chain structure of the physiological breakdown products of the third component of human complement
Authors: Folkersen Jørgen , Teisner Børge , Eggertsen Gösta , Sim Robert B.
Published: May 2005

Immunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified third component of human complement (C3) and its fragments and of unpurified C3 and its degradation fragments in serum or plasma has been examined. The pattern of C3 degradation observed corresponded well with previous observations made with purified proteins. The immunoblotting technique demonstrated a high resolution, and proved to be capable of providing a full analysis of mixtures of C3 fragments in serum with the use of chain‐fragment‐specific antibody preparations. Additional heterogeneity in C3 and its breakdown products were observed, and covalent adducts between activated C3 and other proteins were detected directly in serum and plasma without any purification of the components. The results further indicated that antibodies raised against denatured C3 reacted quantitatively and qualitatively better with the nitrocellulose‐bound C3 than those antibodies raised to native C3, probably reflecting the paratially denatured state of molecules after separation by polyacrylamide gel electrophoresis and blotting onto the nitrocellulose.
A novel method for molecular weight determination in immunoblots
Authors: Hohmann Art , Lemon Jodie , Nikoloutsopoulous Tony
Published: May 2005

A novel method is described to serve as a positive control by including an antiserum raised against and reacted with blots of the molecular standards used in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The marker antiserum may find further application on nylon based adsorptive membranes which cannot be stained by conventional methods.
Galactokinase: Technique for polacrylamide gel isoelectric focusing
Authors: Stambolian Dwight
Published: May 2005

A new technique is presented for the detection of human galactokinase from crude hemolysate by the method of polyacrylamide gel isoelectric focusing. The method utilizes low sample volume and can be completed within 5 h. One gel can be used to screen 15 samples, making the procedure ideal for screening erythrocyte galactokinase in newborn screening programs. This method can be ideal for many laboratories interested in screening for galactokinase variants.
The condensation of ampholytes in steady state moving boundaries: Analysis by computer simulation
Authors: Mosher Richard A. , Thormann Wolfgang
Published: May 2005

A digital simulation of the behavior of amphoteric sample components in moving steady state boundaries is presented. Complete computer simulation data, including profiles of concentration, conductivity and pH as functions of time, are given for both cationic and anionic electrolyte configurations which incorporate one amphoteric sample constituent. The condensation of ampholytes in steady state moving boundaries is shown to proceed via an isotachophoretic mechanism and not by isoelectric focusing. Mobility (velocity) relationships necessary for sample components to form steady state zones are discussed.
Urea‐glycerol‐acrylamide gel electrophoresis of acidic low molecular weight muscle proteins: Rapid determination of myosin light chain phosphorylation in myosin, actomyosin and whole muscle samples
Authors: Sobieszek Apolinary , Jertschin Peter
Published: May 2005

We have improved the Perrie and Perry method (Biochem. J. 1970, 119, 31–38) of urea‐electrophoresis by the addition of a stacking gel containing 8.5 M urea and increased buffer capacity of the glycerol‐containing separation gel. The system has been successfully applied to the analysis of myosin phosphorylation levels not only in myosin and actomyosin, but also in whole muscle samples dissolved in urea. By subjecting the samples to a simple centrifugation and filtration step, all myosin light chains including the unphosphorylated and phosphorylated species as well as calmodulin and troponin could be identified. With a sensitivity of about 5 pmols the procedure described eliminates the need for the time‐consuming two‐dimensional electrophoresis which is commonly used to determine phosphorylation levels in muscle samples.
Efficient protein blotting using an economical power supply
Authors: Fischer Itzhak
Published: May 2005

A simple and inexpensive system of a power supply for protein blotting is described. The blots are run using a 12 V car battery charger which is modified for laboratory use. Under these conditions even high molecular weight proteins of 300 000 can be completely transferred to the nitrocellulose paper in less than 5 h and efficiently immunostained.
A novel method for permanent imprints from dye‐stained electrophoretograms
Authors: Ramasamy Muthiah , Wimmer Dave H. , Kurup Viswanath P.
Published: May 2005

A simple method for obtaining permanent imprints of Coomassie Blue and Amido Black stained electrophoretograms on developed unexposed photographic paper and film is described. Copies of slab and two‐dimensional electrophoretograms and crossed immunoelectrophoretograms were obtained by this technique. The method is fast, simple, inexpensive and can be adapted to make as many as five copies by repeated staining of the electrophoretogram. The copies are the exact size of the original gels and can be stored for a long time. The possible use of these imprints for the comparative study of immunoblots with the corresponding protein‐stained gels is discussed.
Non‐destructive sequential spread tissue isoelectric focusing
Authors: D'Andrea Alberto L. , Torres Leedhamsp VeróNica M.
Published: May 2005

A method of sequential spread tissue isoelectric focusing is described, based on spreading a small amount of defrosted tissue, from musclebiopsy, on a rectangular filter‐paper applicator. A single tissue sample is applied sequentially at six different sites of an agarose gel and after focusing for contact intervals, between gel and applicator, of 15, 5, 5, 5, 5 and 15 min, isoelectric focusing is continued until 1500 Volt × hours are reached. The six patterns observed for a single tissue sample were qualitatively similar and sections of the same gel could thus be used for different detection methods, such as protein stainig, enzyme visualization or immunological detection.
Microscale electrophoresis of stress proteins induced by chemicals during the in vivo cell cycle
Authors: Pipkin James L. , Anson Jeanne F. , Hinson William G. , Robert Burns E. , Casciano Daniel A.
Published: May 2005

The incorporation of leucine and phosphate in proteins extracted with two different solvents from nuclei sorted physically from the G0+G1 phase of the rat submaxillary gland cell cycle was observed by two‐dimensional microscale polyacrylamide gel electrophoresis. Sodium phenobarbital did not initiate additional isotope ([3H]leucine and [32P]phosphate) incorporation on its own. After dosing with DL‐isoproterenol, several proteins incorporated leucine and phosphate during the G0+G1 phase. The inclusion of phenobarbital in the dosing regime enhanced labeling of several proteins already present in the G0+G1 phase. Treatment of animals with sodium arsenite prompted labeling of phosphate of two “stress” proteins in the non‐dividing G0 phase of the submaxillary gland. Enhanced incorporation of proteins was exhibited after combined dosing of isoproterenol and sodium arsenite, but no new incorporated proteins were seen in the G0+G1 phase. Likewise, no new proteins were seen to incorporate in the G2+M phase, but there was some increased incorporation in a few already present proteins under these conditions. The similar mobilities of several stress proteins, which were determined by coelectrophoresis of stress marker proteins, suggested that several of these proteins, even though they were extracted with different solvents, were identical.
Multiple forms of serum aminopeptidases separated by micro two‐dimensional electrophoresis under non‐denaturing conditions
Authors: Sanderink Ger‐Jan C. M. , Artur Yves , Galteau Marie‐Madeleine , Wellman‐Bednawska Maria , Siest Gérard
Published: May 2005

Micro two‐dimensional electrophoresis in the absence of denaturing agents allowed the analysis of the multiple forms of serum aminopeptidases, including their multimolecular complexes, and the detection of their enzymatic activity towards different substrates. Two forms of alanine aminopeptidase (E.C. 3.4.11.2.), differing in sialic acid content, were detected in sera of healthy subjects. In sera of patients with hepatobiliary diseases a large number of additional spots was observed, some of them identified as multimolecular complexes, probably formed by an amphiphilic form of alanine aminopeptidase. Papain treatment resulted in the dissociation of the complexes and in the appearance of a light form of the enzyme in normal sera, which was otherwise only present in pathological samples. Additional spots corresponding to cysteine aminopeptidase (E.C. 3.4.11.3.) were detected in pregnancy sera.
Protein staining by Coomassie Brilliant Blue G‐250: Unique color rendering in iron‐toned prints
Authors: Sippel Theodore O.
Published: May 2005

Black‐and‐white photographs of protein staining by Coomassie Brilliant Blue G‐250 may be iron‐toned by a modified procedure to obtain good reproduction of the original hue and range of intensities. A suitable paper and a film for transparencies are specified for printing from a given negative.
Computer simulation of two electrophoretic columns coupled for isoelectric focusing in simple buffers
Authors: Tsai Amos , Mosher Richard A. , Bier Milan
Published: May 2005

Computer simulation is used to analyze a system of two electrophoretic columns coupled by mixing the anolyte of one with the catholyte of the other. A mathematical model is presented which is used to predict the pH gradients formed by monovalent buffers in this system, when the currents in the columns are unequal. In the column with the higher current a pH gradient is created which increases from anode to cathode and is potentially useful for isoelectric focusing. The breadth of this gradient is dependent upon the ratio of the currents. The function of the second column is the compensation of buffer migration which occurs in the first column, thereby maintaining constant electrolyte composition. The effects of buffer pKs and mobilities are evaluated.
High resolution two‐dimensional electrophoresis of hair proteins without prior S‐carboxymethylation
Authors: Marshall Thomas , Williams Katherine M.
Published: May 2005

Hair proteins have been successfully analysed by high resolution two‐dimensional electrophoresis by avoiding S‐carboxymethylation which has previously dictated the use of alternative two‐dimensional procedures. The high and low‐sulfur proteins are simultaneously resolved within the pI range 3–9 and their electrophoretically determined Mr values are consistent with molecular weights obtained by physical methods.
The use of polyacrylamide gels that can be dissassembled and reassembled to permit a wide variety of electrophoretic procedures with a common polymerization history
Authors: Horowitz Paul , Bowman Steven
Published: May 2005

A demountable polyacrylamide gel system is described that uses commercial components and can be adapted to any slab system. The use of GeldBond PAG film produces polyacrylamide gels that can be easily handled and allows separation of the conditions needed for polymerization from those for electrophoresis. Polymerization is performed with one common protocol, and the gel is disassembled. Gels can then be soaked or stored in any desired solutions, followed by reassembly of the equilibrated gel and electrophoresis. This procedure allows for a wide range of electrophoretic conditions starting with a single type of polymerized gel as well as permitting use of systems that have technical difficulties under normal cirumstances such as horizontal gels or those containing the cationic detergent, cetyltrimethylammonium bromide.
Application of recycling isoelectric focusing for purification of recombinant human leukocyte interferons
Authors: Nagabhushan Tattanahalli L. , Sharma Basant , Trotta Paul P.
Published: May 2005

A computer‐controlled version of recycling isoelectric focusing has been applied to the purification of recombinant human leukocyte interferons (IFNs) from E. coli. The system is based on continuous recycling between a 12‐channel heat‐exchange reservoir and a 12‐channel focusing cell. It contains ten pH meters with probes in each of the flowing channels and two ultraviolet spectrophotometers with five flow cells per instrument. A minicomputer system acquires real‐time data of pH and optical density measurements, controls and continuously monitors voltage and power outputs across the focusing cell, and generates on‐line multi‐color graphics on an x‐y plotter. Recycling isoelectric focusing in the presence of 4 M urea resulted in removal of contaminating bacterial pyrogens from recombinant IFN alpha‐2 with complete retention of specific antiviral activity. It also produced a 25‐fold purification of crude recombinant IFN alpha‐5 in a single step and resolved two biologically active species. These data suggest that recycling isoelectric focusing may be incorporated into the design of simple schemes for the purification of a variety of recombinant proteins.
Affinophoresis of anti‐hapten antibody in rabbit serum with an anionic affinophore bearing the hapten
Authors: Shimura Kiyohito , Kasai Ken‐ichi
Published: May 2005

Affinophoresis is an electrophoretic separation technique for biomolecules using an affinophore, which is a macromolecular polyelectrolyte bearing affinity ligands. It migrates rapidly in an electric field, and consequently the electrophoretic mobility of molecules having affinity for the ligand is specifically changed. An anionic affinophore bearing an antihypertensivepeptide, N‐(dibenzyloxyphosphinoyl)‐L‐alanyl‐L‐prolyl‐L‐proline, was synthesized by coupling the peptide to about one‐sixth of the amino groups of poly‐L‐lysine with an average degree of polymerization of 190. The remaining amino groups were succinylated and aminomethanesulfonic acid was coupled to about one‐fourth of the succinyl groups by the formation of amide bonds. Electrophoresis of rabbit antiser a raised against the peptide was carried out in an agarosegel plate containing the affinophore. After electrophoresis, separated proteins were transferred to a nitrocellulose sheet and immunoglobulins were visualized by using horseradish peroxidase‐conjugated anti‐rabbit immunoglobulin G antibody. Hapten‐specific antibodies were separated from non‐specific antibodies. Two‐dimensional agarose gel electrophoresis, in which affinophoresis was used in the second dimension, separated the hapten‐specific antibodies from other serum proteins.
A simplification of Heukeshoven and Dernick's silver staining of proteins
Authors: Damerval Catherine , Guilloux Martine Le , Blaisonneau Joël , de Vienne Dominique
Published: May 2005

The procedure of Heukeshoven and Dernick for silver staining of proteins in polyacrylamide gels is simplified by applying Farmer's redücer before impregnation with silver nitrate and rinsing the gel in 2.5 % sodium carbonate instead of distilled water. This modification yields pattern of good contrast without a destaining‐restaining step.
Separation of soybean leghemoglobin components by isoelectric focusing in immobilized pH gradients
Authors: Puppo Alain , Rigaud Jean
Published: May 2005

Soybean leghemoglobin subcomponents were separated with good resolution in immobilized pH gradients, both at the analytical and preparative scales. In the latter case, the tedious step of gel washing could be omitted without affecting resolution. This procedure appeared as the only one suitable for preparing large amounts of highly purified leghemoglobin c1, c2 and c3, allowing their further characterization and an investigation of their specific roles in nitrogen fixation.
Human transcobalamin isopeptides: Separation by isoelectric focusing and by polyacrylamide gel electrophoresis
Authors: Hansen Mads , Fràter‐Schröder Marijke
Published: May 2005

Transcobalamin was separated by isoelectric focusing and by polyacrylamide gel electrophoresis. Isoelectric focusing showed one band in homozygotes and two bands in heterozygotes, with isoelectric points between 6.3 and 7.0, while polyacrylamide gel electrophoresis produced two and four bands, respectively. Addition of neuraminidase, urea or EDTA had no effect on the separation of transcobalamin at isoelectric focusing. The results obtained by the two methods are comparable and in accord with the nomenclature of transcobalamin variants.
Determination of cell mixtures by an automated cell electrophoretic instrument and monoclonal antibody
Authors: Hayashi Haruhisa , Toyama Naomi , Fujii Masahiko , Yoshikumi Chikao , Kawai Yoshio , Iwaguchi Takao
Published: May 2005

An automated cell electrophoretic instrument, Parmoquant‐L, is based on the opto‐electronic image analysis whose principle is the same as conventional cytopherometers. Experiments are performed with high precision (C.V. values < 1.5 %) and in short time intervals of 5 min. Using this instrument the cell populations of a mixtureof sheep red blood cells (SRBC) and rabbit red blood cells (RRBC) were determined. The ratio of high mobility cells (HMC) to low mobility cells (LMC) was identical with the mixed ratio of SRBC to RRBC (R = 0.998) and there was no influence of different cell diameters between the two populations. As the electrophoretic mobilities of mouse red blood cells (MRBC) and SRBC were almost the same, the mixture of the two populations showed a broad unimodal electrophoretic mobility pattern. After the mixture was incubated with anti‐SRBC monoclonal antibody (diluted to 1/100), the mobility of SRBC decreased 30 % and it was possible to determine the cell numbers of each erythrocyte in the mixture. From these results, Parmoquant‐L is suitable to determine the populations in the mixture of different erythrocytes.
Two‐dimensional electrophoresis with immobilized pH gradients in the first dimension: Protein focusing as a function of time
Authors: Hanash Samir M. , Strahler John R. , Somerlot Luke , Postel Wilhelm , Görg Angelika
Published: May 2005

Since the introduction of immobilized pH gradient (IPG) electrophoresis, optimization of sample entry has been a major concern. Partial sample entry, associated with smearing of proteins, has led investigators to propose the use of hybrid IPG‐carrier ampholyte focusing for adequate protein separation. We have examined the effects of adding carrier ampholytes to the protein sample, preelectrophoresis and the site of sample application on sample entry, resolution and pattern stability for IPG gels, used as a part of two‐dimensional electrophoresis. The addition of carrier ampholytes to the sample increased the rate of entry of proteins into the gel while preelectrophoresis had an opposite effect. There was a substantial difference in protein entry between sample application at the anode vs. cathode. After an adequate focusing time the final focusing pattern was remarkably similar and consisted of several hundred well‐resolved polypeptides, irrespective of the initial starting conditions. Therefore IPG electrophoresis, without addition of carrier ampholytes to the gel, results in the high resolution separation of complex protein mixtures.
A modified applicator strip for improved focusing on immobilized pH gradient gels
Authors: Pflug Werner , Laczko Bela
Published: May 2005

The use of applicator strips on isoelectric focusing in immobilized pH gradient gels often results in lateral sample spreading and mixing of adjacent samples. Both effects can be reduced or eliminated if a modified applicator strip with slits at the bottom is used. Different modes of sample application were compared using the group specific component system with different concentrations of bovine serum albumin in the samples.
Reduced area two‐dimensional gels for direct digital imaging of radioactive protein profiles
Authors: Ross Mary E. , Latter Gerald , Burbeck Stephen , Leavitt John
Published: May 2005

A method is demonstrated which reduces the area of a standard two‐dimensional polyacrylamide electrophoresis gel to approximately one‐fourth of its original area. Microdensitometry is used to show that the technique did not significantly alter the reproducibility of the gels. This reduction in size makes it possible to use a micro‐channel plate analyzer to directly digitize an entire gel.
Moving boundary electrophoresis on agarose gel of plant viruses and polystyrene microspheres
Authors: Gombocz Erich , Tietz Dietmar , Chrambach Andreas
Published: May 2005

Three plant viruses: turnip crinkle (TCV), hibiscus chlorotic ringspot (HCRSV) and pelargonium flowerbreak (PFBV), and polystyrene size standards with radii of 22.4–59.4 nm can be stacked within trailing and leading ion net mobilities of 0.059 to 0.273 (relative to Na+). Stacking was carried out at pH 6.50, 0.03 M ionic strength, 50 mM 3–[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate, at a gel concentration of 0.4 %, in agarose gel electrophoresis conducted at 1.2 mA/cm2 of gel. Unstacking occurs under the same conditions at gel concentrations ranging from 0.5 to 1.1 % agarose, while it can be brought about between 0.1 and 0.7 % agarose when the pH is raised to 7.27, corresponding to a front moving boundary with a trailing ion net mobility of 0.216 (relative to Na+). Ferguson plots of viruses and polystyrene particles in the discontinuous buffer system are curvilinear and comparable to those obtained in a continuous buffer at pH 6.50 of the same composition and operative pH as that of the resolving phase of the discontinuous buffer. Particle radii and net charge values can be obtained from the non‐linear Ferguson plot in the discontinuous buffer system by previously reported methods of computer simulation, but this Ferguson plot presents a more limited data base than that in the continuous buffer since it excludes gel concentrations which yield relative mobility (Rf) values of 1.0. Since computer simulation provides the range of gel concentrations in which both the fiber radius and length [1], as well as the size of the particle [2], remain constant for a particular preparation of agarose, a simplified alternative method of evaluating particle sizes exists. Within that specific gel concentration range, the linear segment of the Ferguson plot can be used to compute particle sizes by an operationally convenient, albeit approximative, method, using the same PAGE‐PACK programs of D. Rodbard which are commonly used in the size determination of macromolecules by polyacrylamide gel electrophoresis. The two methods of particle size determination, based on either the entire non‐linear Ferguson plot or on its linear segment in the appropriate gel concentration range, yield similar results (average deviation 12 %). The radii of three plant viruses are dependent to different degrees on the presence of Ca++ in the electrophoretic system. Values obtained in the presence of Ca++ are comparable to those found by electron microscopy.
Affinity electrophoresis with monoclonal antibodies
Authors: Breborowicz Jan , Gan Jacek , Klosin Jadwiga , Gryska Katarzyna , Majewski Przemyslaw , Breborowicz Danuta , Waliszewski Przemyslaw
Published: May 2005

Affinity electrophoresis was applied to the study of binding of monoclonal antibodies (MoAb's) with their respective antigens. The MoAb's may be either mixed with the antigen in an application well or incorporated into the agarose gel. The first approach is more convenient for studying high affinity MoAb's, while MoAb's with low affinity might be more efficiently studied in the second system. Our conditions for affinity electrophoresis do not imply nor necessitate immobilization of either an antigen or MoAb and are most suitable for studies of MoAb's reacting with antigens of alpha mobility demonstrated in experiments with MoAb's against human alpha‐fetoprotein (AFP). Both AFP‐MoAb and AFP‐MoAb‐AFP complexes were observed during electrophoresis. The identity of these complexes was confirmed by their reaction both with polyclonal antibodies against mouse immunoglobulins and against human AFP. The molecular weight of complexes was determined by gel filtration. Affinity electrophoresis was less convenient for studies of MoAb's reacting with antigens of gamma mobility at pH 8.6. Nevertheless, MoAb's against carcinomembryonic antigen, human IgG and human IgA did bind with their respective antigens under the conditions of affinity electrophoresis. The binding of MoAb's with these antigens during affinity electrophoresis was confirmed by demonstration of mouse immunoglobulins in the precipitates formed by reaction of the studied antigens with polyclonal antibodies. Our results point out the suitability of affinity electrophoresis for studies of MoAb's.
Fast preparative isoelectric focusing of phycocyanin subunits in layers of granulated gels
Authors: Köst‐Reyes Eliana , Schneider Siegfried , John Wolfhart , Fischer Richard , Scheer Hugo , Köst Hans‐Peter
Published: May 2005

A new method is presented for the fast preparative separation of the light‐harvesting photosynthetic pigment C‐phycocanin into its α and β subunits, which is based on isoelectric focusing in layers of granutaled gels containing 7 M urea. The method has been successful in cases where other separation procedures failed. The recovery of the separated chains of the light ‐sensitive biliprotein amounts to 70 ± 10 % when the separation is carried out under light exclusion and in an argon atmosphere. A simple and inexpensive setup for work under an atmosphere of protective gas is described.