bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of DNA superhelix density by dye titrationslab gel electrophoresis
Authors: Poddar Saibal K. , Maniloff Jack
Published: May 2005

An ethidium bromide titration procedure using electrophoresis in horizontal multigels is described which facilitates measurement of DNA superhelix densities. Using this system, measured superhelix density was found to be dependent on the amount of DNA loaded on the gel.
Easy “home‐made” immobilized pH gradients forisoelectric focusing
Authors: Charlionet Roland , Sesboüé Richard , Davrinche Christian
Published: May 2005

Acrylic acid, methacrylic acid, itaconic acid and dimethylaminopropylmethacrylamide can easily be introduced in polyacrylamide gels in order to obtain reproducible immobilized pH gradients in the pH range 4–6. These acrylic derivatives are commercially available at a relatively low price.
The influence of mercaptoethanol on the apparent molecular weights of jojoba and standard proteins separated by porosity gradient gel electrophoresis in presence of urea
Authors: Shah Akbar A.
Published: May 2005

Proteins exhibit higher apparent molecular weights when treated with mercaptoethanol and separated electropretically in a porosity gradient in presence of ureacompared to their apparent molecular weights without mercaptoethanol. This unexpected effect was first noticed with proteins of jojoba. The apparent increase was also seen when oligomers of serum albumin were separated, but was absent with gelatin, indicating a reaction of disulfide groups in the polypeptide chain of serum albumin. When the proteins were treated with hydrogen peroxide prior to electrophoresis the apparent increase of molecular weights of jojoba proteins after mercaptoethanol treatment was less evident in the presence of monomethylurea and was absent in gels containing tetramethylurea.
Rapid detection and characterization of plant virused by agarose gel electrophoresis: Size, surface charge and heterogeneity of panicum mosaic and related viruses
Authors: Serwer Philip , Moreno Elena T. , Hayes Shirley J. , Berger Philip , Langham Marie , Toler Robert W.
Published: May 2005

Using panicum mosaic virus (PMV) and several St. Augustine decline strains of PMV (SAD) as test viruses, previously developed procedures of agarose gel electrophoresis (reviewed in P. Serwer, Electrophoresis 1983, 3, 375–382) have been used for the first time to detect and characterize plant viruses. PMV and some SAD strains produce two particles that copurify during rate zonal sedimentation, but form separate bands during agarose gel electrophoresis (forms 1 and 2). By measuring electrophoretic mobility (μ) as a function of agarose percentage and correcting for electroosmosis, it was found that: (a) form 1 has a solid support‐free μ (μ0) different from the μ0 of form 2, and (b) forms 1 and 2 have the same radius (14.6 ± 0.6nm). Form 1 is indistinguishable from form 2 by either electron microscopy or sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The μ0 of form 1 and the relative amount of forms 1 and 2 vary with the virus strain and can be used to help identify strains. A previously described satellite of PMV and some SAD strains also has two forms. The radius of the satellite of one SAD strain was found to be 7.6 ± 0.3 nm. A procedure of differential gel staining was developed for distinguishing the 14.6 nm virus from its 7.6 nm satellite. After agarose gel electrophoresis of either totally unfractionated or partially fractionated, infected grass extracts, virus particles wer detected without interference from host components. The procedures used here should be useful for rapid disease diagnosis and strain typing of the above and possibly other plant viruses.
Glycosylation of human abumin in diabetes mellitus: Extensive microheterogeneity of serum and urinary species as revealed by isoelectric focusing
Authors: Candiano Giovanni , Ghiggeri Gian Marco , Delfino Gerolamo , Queirolo Carlo , Gianazza Elisabetta , Righetti Pier Giorgio
Published: May 2005

Albumin and glycosylated albumin from serum and urine of 10 diabetic patients have been studied by ultrathin‐layer isoelectric focusing andsilver staining. Both total serum albumin and its glycosylated form were microheterogeneous with a main band focused in the pH 4–5 range; in the case of glycosylated ablumin several cathodic bands with pI's up to 7 were found to form a discretely abundant population among the total species. Some similarites and some striking differences were observed for urinary albumin, which, beside the main unmodified albumin with pI ca. 4.8, was enriched in microheteregeneous more anodal bands (PI 4.0–4.7 species). All these anodal bands were tightly bound by concanavalin A‐Sepharose, suggesting their identity as glycosylatedalbumin. these observations indicate that the glycosylation of albumin is not an equimolar reaction, but it can involve many sites of attachement of carbohydrates. The also suggest that albumin species with higher net negative charge and higher content of carbohydrates are preferentially able to pass through the glomerular barrier, providing the descriptive basis of diabetic albuminuria. Their relevance to diabetic nephropathy is a goal for further work.
Double‐label autoradiography revisited: II. The 35S and 75Se‐system
Authors: McConkey Edwin H. , Anderson Catherine
Published: May 2005

The use of 75Se in combination with 35S for double‐label autoradiography has been quantitated. For gels 0.8 mm thick impregnated with EN3HANCE, the input ratio of 75Se dpm/35S dpm should be 3.3; and the second stage autoradiogram, if made with an interposed sheet of blackened XAR films as a screen to block 35S, should be exposed 7.8x as long as the flourogram for SB film or 5.6 × as long as the fluorogram for XAR film. However, autoradiograms made under these conditions have larger, more diffuse spots than those made with the 3H/35S system and the cost of [75Se]selenomethionine is much higher than the cost of [3H]methionine.
Separation of the primary isoenzyme bands (a±, b±) determined by the phosphoglucomutase (PGM1) locus on an immobilized pH gradient
Authors: Sutton John G. , Westwood Sara A.
Published: May 2005

This report describes a method of typing the phosphoglucomutase (PGM1) locus (EC 2.7.5.1) isoenzymes from bloodstains on an immobilized pH gradient. A pH 5.8 to 6.8 gradient was prepared in which only the primary typing bands, a± and b±, associated with the 10 common PGM1 locus phenotypes were separated. This system provides the forensic scientist with a method of typing anomalous results where confusion has arisen over the identity of the b+ and the c−/c+ isoenzymes observed by conventional electrofocusing. Successful typing of 2‐month‐old bloodstains was achieved within 2.0 h.
Rapid electrofocusing of erythrocyte acid phosphatase
Authors: Budowle Bruce
Published: May 2005

A routine method is described for obtaining reproducible erythrocyte acid phosphatase patterns by ultrathin‐layer polyacrylamide gel isoelectric focusing. Electrofocusing of EAP on 0.2 mm thick gels took 30 and 22 min on the Ultrophor and Cold Focus Apparatus, respectively, when the distance separating the electrodes was 5.4 cm. This method, which reduces the cost for focusing EAP, can be ideal for many laboratories interested in EAP phenotyping.
pH gradient characteristics of commercially available carrier ampholytes and their uses for typing the human phosphoglucomutase locus (PGM1)
Authors: Gill Peter , Sutton John G.
Published: May 2005

A number of different carrier ampholyte mixtures were compared in order to produce a pH gradient which separated the phosphoglucomutase‐1 (PGM1) phenotypes most efficiently. Ultrathin gels incorporating a mixture of pH 5–7 and pH 6–8 Ampholine was shown to produce the best results. Application of samples to the cathodal end of the gel reduced the problem of overloading which is frequently encountered with ultrathin gels.
Esterase D: Simultaneous electrophoretic determination of the three common allozymes (ESD 1, 2, 5)
Authors: Bär Walter , Häni Markus , Biedermann Verena
Published: May 2005

A simple and time‐saving electrophoretic method for the determination of the 6 common phenotypes of red cell esterase D (ESD) is presented. Using a continuous buffer system composed of malic acid, the separation of the allozymes is accomplished at pH‐values between 5.35–5.45 by thin‐layer agarose gel electrophoresis in 4 h. Until now, a combination of electrophoresis and isoelectric focusing was required to obtain the same results. The economical advantage as well as the modest requirements concerning laboratory equipment are evident.
Analysis of free zone electrophoresis of fixed erythrocytes performed in microgravity
Authors: Snyder Robert S. , Rhodes Percy H. , Herren Blair J. , Miller Teresa Y. , Seaman Geoffrey V. F. , Todd Paul , Elaine Kunze M. , Sarnoff Burton E.
Published: May 2005

A free fluid zone electrophoresis experiment was performed in the microgravity environment of Space Shuttle flight STS‐3 (March 1983). The experiment was designed to confirm observations made on the Apollo‐Soyuz mission of 1975 and to test the effect of high red blood cell (RBC) concentration on free fluid electrophoresis. Photographic documentation of cell zone progression in one‐hour separations of mixtures of formaldehyde‐fixed human and rabbit erythrocytes (RBC), which were subjected to a field of approximately 13 V cm−1 in low ionic strength buffer, was analyzed. One of two columns contained 2 × 108 RBC ml−1 (low concentration), and the other contained 1 × 109 RBC ml−1 (high concentration). The observed and calculated leading edge displacements of the RBC in the two columns were in agreement, indicating the absence of unexpected effects of the reduced gravity environment. Post‐flight analyses of the contents of the columns was not possible, and additional microgravity experiments are needed to evaluate the role of particle‐particle interactions in concentrated suspensions undergoing electrophoresis.
Preparative isoelectric focusing in immobilized pH gradients IV. Recovery of proteins from Immobiline matrices into ion‐exchange resins
Authors: Casero Patrizia , Gelfi Cecilia , Righetti Pier Giorgio
Published: May 2005

A new electrophoretic transfer system for the recovery of proteins focused in Immobiline matrices is described. The gel strip containing the sample zone of interest is transferred to a horizontal tray and embedded in 1%, low‐gelling (37°C) agarose. For acidic to neutral proteins (up to pI 7.7) the electrophoretic transfer is from the IPG strip into a layer of DEAE‐Sephadex, buffered at pH 8.5 in 100 mM Tris‐acetate. Recovery (better than 90% in all cases studied) is achieved by titrating the resin at pH 9.5, in 200 mM Tris‐Gly buffer, containing 200 mM salt. For basic proteins (pI>7.7) the electrophoretic retrieval is from the IPG strip into a zone of CM‐Sephadex, buffered at pH 6.0, in 50 mM citrate (cathodic migration). Recovery (again better than 90%) is accomplished by titrating the exchanger at pH 4.0, in 200 mM formate buffer, containing 200 mM NaCl. Exposure to pH 9.5 does not affect enzyme activity if the eluate is promptly titrated around neutrality; a pH 4 milieu might irreversibly alter enzymes, in which case elution is best performed by titrating the protein to its pI, rather than protonating the exchanger. It has been demonstrated that Immobiline gels, even when incorporating 5 times the standard amount of buffer (75 mM Immobiline at pH = pK, i. e. 50 mM buffering ion and 25 mM titrant) exhibit, under the electric field, negligible ion‐exchange properties, thus ensuring ideal behavior as supports for isoelectric focusing.
Computer‐aided analysis of the electrophoretic regulating function omega
Authors: Thormann Wolfgang , Mosher Richard A. , Bier Milan
Published: May 2005

The omega function, defined for strong and monovalent weak electrolytes, is a useful regulating principle for the description of electrophoretic processes. The omega function profile, which is established by the initial electrolyte distribution in an electrophoretic column, will not be changed by current flow. The function is based solely upon migration and is only applicable to those regions where diffusional dispersion can be neglected. Computer simulations are used to follow the evolution of the concentration gradients of various electrophoretic systems and to verify the proposed constancy of the omega function profile. The limitations of the theory are investigated and the value of these predictions to the experimentalist is discussed.
A two‐dimensional electrophoretic profile for bacterial esterases
Authors: Goullet Philippe , Picard Bertrand
Published: May 2005

Esterases produced by different bacterial species were separated by conventional electrophoresis (CE) in polyacrylamide agarose gel and by thin‐layer isoelectric focusing (IEF). Although CE revealed the greatest number of esterases and was preferable for their identification by specific hydrolytic activities, the two techniques appeared to be complementary in their resolving power for detection of electrophoretic variants. Consequently, by establishing a direct correspondence between homologous esterase bands resolved by CE and IEF, we have proposed a two‐dimensional electrophoretic profile (2‐DEP) which considerably refined the degree of esterase polymorphism and improved the enzymic differentiation between and within bacterial species.
Determination of amprolium in feed stuff by capiliary isotachophoresis
Authors: Křivánková Ludmila , Boček Petr
Published: May 2005

A method for the determination of amprolium in feed stuff by analytical capillary isotachophoresis with column coupling is described. Up to 20 μl of the raw water extract of amprolium from a feed stuff can be analysed directly without any pretreatment in 15 min. The detection limit reached the level of ppm, the reproducibility of analyses in the followed range of 0.2–1.2 nmoles was 3 rel. %.
Two‐dimensional gel electrophoresis of low mobility group non‐histone proteins from myeloma cells
Authors: Bouchonneau Michel , Durand Jean Pierre , London Jacqueline , Pieri Jacques
Published: May 2005

Non‐histone proteins (NHP) extracted from chromatin of two plasmocytoma cell lines, X63‐Ag8‐653 and Sp2/O‐Ag 14, with 0.35 M NaCl are analyzed by two‐dimensional polyacrylamide gel electrophoresis. These NHP have been fractionated into a high mobility group (HMG) and a low mobility group (LMG) using 2 % trichloroacetic acid (TCA). The HMG which is well‐characterized is soluble in 2 % TCA whereas the LMG precipitates. LMG resolubilization is difficult. Only a buffer containing glycerol, sodium dodecyl sulfate (SDS) and β‐mercaptoethanol enables them to be studied by polyacrylamide SDS electrophoresis. The LMG is clearly differentiated from the HMG by the predominance of high molecular weight proteins and by the high number of molecular species in a wide pH range. In the LMG there are also some diffrences between the two plasmocytoma cell lines. For example, a high molecular weight protein of 100 000 and pI 7.2–8.2 is present in Sp2/O‐Ag 14 but absent in X63‐Ag8–653. Also noticeable in the LMG proteins is the presence of low molecular weight proteins with high electrophoretic mobility.
Investigation of the stability of anionic isotachophoretic zones formed by ethylenediaminetetraacetic acid and nitrilotriacetic acid metal complexes
Authors: Thormann Wolfgang
Published: May 2005

The formation of anionic complexes with chelating ligands like ethylenediaminetetraacetic acid or nitrilotriacetic acid is useful for the anionic isotachophoretic determination of selected metals. A zone comprising such a metal complex may decompose gradually under the influence of the electric current. The kinetic behavior of various complex zones is determined in a capillary‐type apparatus with a linear array of 255 potential gradient sensors. The discussed examples comprise both systems where complex formation occurred in the sample solution prior to its injection into the isotachophoretic column and where an in situ formation of the complex took place under current flow.
Application of separator isoelectric focusing within the pH range 4‐6
Authors: Gill Peter
Published: May 2005

Separator isoelectric focusing is a simple technique for creating narrow pH gradients (less than 1 pH unit wide). It has proved useful elsewhere for the enhanced separation of phosphoglucomutase, esterase D, group specific component (Gc) haemoglobin, transferrin and for distinguishing between seminal and vaginal acid phosphatases. The properties of 10 different zwitterionic buffers have been studied in order to determine pH gradient profiles, their stability, experimental reproducibility and the effect of temperature. The effect of adding non‐ionic constituents (glycerol or sucrose) to the gel has also been investigated. The buffers examined produced narrow pH gradients within the pH 4–6 range. It was shown that specific pH gradients could be produced by mixing different buffers and carrier ampholytes together.
Negative aurodye for polyacrylamide gels: The impossible stain
Authors: Casero Patriza , del Campo Gian Battista , Righetti Pier Giorgio
Published: May 2005

A negative aurodye for polyacrylamide gels has been developed. After isoelectric focusing in 6 % T polyacrylamide gels, protein fixation and washing, the gel is immersed in 0.01 % HAuCl4 containing 0.19 % citric acid and 0.1 % Tween 20, freshly made. The free reactants diffuse within the gel fibers and, upon micelle formation, stain the gel matrix purple, leaving white protein zones in sharp contrast. The sensitivity is of the order of 15–20 ng protein/mm2, matching and even passing that of silver stain (the lowest detection limit of Coomassie Blue being around 300 ng protein/mm2). The mechanism of background stain does not seem to depend on hydrophobic interaction with the gold‐detergent micelle, as polyacrylamide gelled with hydrophobic cross‐linkers has markedly decreased stain‐binding ability. The formation of mixed micelles of gold with non‐ionic, zwitterionic and anionic detergents has been studied.
Charge heterogeneity of human protein C revealed by isoelectric focusing in immobilized pH gradients
Authors: Gelfi Cecilia , Righetti Pier Giorgio , Mannucci Pier Mannuccio
Published: May 2005

Protein C (PC), a plasma zymogen of a serine amidase with strong anticoagulant activity, has been purified to homogeneity as shown by sodium dodecyl sulfate electrophoresis. After isoelectric focusing in immobilized pH gradients, PC consists of six isobands, with isoelectric points (pI) between pH 4.8 and 5.1. That all the isobands were PC was confirmed by electroblotting on cellulose nitrate followed by immunoperoxidase staining. Activated PC gives a similar pattern of bands, with pIs ca. 0.5 units higher. Activated PC was also recognized by the monospecific antibody.
A new approach to crossed immunoelectrofocusing using polyacrylamide gel cylinders and agarose gel cassettes
Authors: Marshall Thomas
Published: May 2005

A new approach for crossed immunoelectrofocusing is described. It combines isoelectric focusing in polyacrylamide gel cylinders and immunoelectrophoresis through vertical antibody‐containing agarose gels enclosed in glass cassettes. The approach, using the same apparatus as for high resolution two‐dimensional electrophoresis, has been successfully applied to human serum proteins and the effect of incorporating an antibody concentration gradient in the immunoelectrophoresis gels demonstrated.
Subtypes of the human complement component C3 revealed by isoelectric focusing in immobilized pH gradients
Authors: Charlionet Roland , Davrinche Christian , Rivat Claude
Published: May 2005

The phenotyping of the third component (C3) of human complement has been performed by isoelectric focusing in immobilized pH gradients followed by immunoblotting on nitrocellulose filter membrane. This powerful technique reveals variations of C3* and C3*F alleles not detected by agarose electrophoresis. The limits of the resolving power of isoelectric focusing in immobilized pH gradients for C3 analysis are shown to depend upon the high molecular weight of this protein. The notion of “suptypes” is discussed. Finally, the importance of subtyping for medical applications and for determination at the molecular level of interacting protein mechanisms is underlined.
The application of spread tissue isoelectric focusing for species' determination of raw muscle tissues
Authors: D'Andreay Alberto L. , Torres Leedham Verónica M.
Published: May 2005

A routine method for species' determination of raw muscle tissues is described, Defrosted raw muscle tissues were evenly spread on filter paper applicators which were then placed on a thin‐layer agarose plate. A pH 4–8 gradient was obtained by mixing equal parts of Ampholine 4–6 and 6–8. Tissue homogenates of bovine, equine, porcine, ovine and other species were compared with spread tissue applications. The proposed method is useful in the routine control for quick identification of cut meat in cases of fraud where an exchange of species may have taken place.
Concentration of proteins in the free‐flow electrophoretic apparatus
Authors: Shukun Sergey A. , Gavryushkin Alexander V. , Brezgunov Vyacheslav N. , Zav'yalov Vladimir P.
Published: May 2005

Protein concentration using step pH and conductivity gradients in a free‐flow electrophoretic apparatus is described. The experiments were carried out in a vertical planar 36.5 × 5.0 × 0.05 cm separation chamber with 48 channels both at the inlet and outlet. The gradients were generated by injecting a 2.5 mM borax solution (pH 9.2, conductivity = 0.5 mS) into the chamber via channels No. 1–12 counting from the cathode, and 0.45 M boric acid (pH 4.3, conductivity = 0.025 mS) together with the protein to be concentrated via the remaining channels. The gradients were suitable for the concentration of the proteins with isoelectric points ≥ pH 7.0 (human immunoglobulin G, bovine hemoglobin). From mixtures of hemoglobin and albumin, the latter was removed during concentration. The protein concentration of the starting solution does not affect the results of concentration. At constant current of 30 mA and a residence time of 45 s, the proteins were concentrated at a rate of 400 ml of starting solution per hour with a maximum concentration factor of 10.
Studies on the electrophoretic separation of the four common human phosphoglucomutase (PGM1) allozymes
Authors: Dissing Jørgen , Bär Walter
Published: May 2005

The electrophoretic properties of the four phosphoglucomutase (PGM1) allozymes were studied in the presence of different buffer constituents at pH values from 4.6 to 6.0. A separation of the allozymes allowing the identification of the 10 common phenotypes was obtained in the entire pH range. The separation of the “1+” and “1−” allozymes and of the “2+” and “2−” allozymes was poor near pH 6 but improved considerably at more acidic pH values. This indicated that the charge difference between the “1+” and the “1−” allozymes and between the “2+” and “2−” allozymes is due to substitution of a neutral amino acid in the “1+” and “2+” allozymes with histidine in the “1−” and “2−” allozymes. Above pH 5 the sequence from cathode to anode was “1−”,“1+”,“2−”,“2+” and below pH 5 it was “1−”,“2−”,“1+”,“2+”. At the lower pH values the separation between the “1−” and the “2−” allozymes and between the “1+” and the “2+” allozymes diminished. Hence only two PGM1 species may exist at more acidic pH (pH« 4.6): a “+” enzyme and a “−” enzyme. The presence of di‐ and polyvalent carboxylic acids in the electrophoretic buffer reduced the mobility of the enzymes towards the cathode or changed it into a migration towards the anode. This “buffer effect” was greatest with the polyvalent carboxylic acids. It is suggested that it is caused by an adsorption of carboxylate ions onto the enzyme surface, which results in a transfer of negative charges to the enzyme. The “buffer effect” varied slightly from one enzyme species to another, and this may explain the general experience that the electrophoretic resolution of a protein mixture differs when different buffer systems are used in the electrophoretic medium.
Improved horizontal two‐dimensional electrophoresis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying‐on transfer to the second dimension
Authors: Görg Angelika , Postel Wilhelm , Günther Siegfried , Weser Johann
Published: May 2005

Two‐dimensional (2‐D) maps obtained with horizontal or vertical electrophoretic systems are compared. There are no differences in resolution, spot size and distribution, but significant differences in handling, Horizontal 2‐D electrophoresis, where the equilibrated isoelectric focusing (IEF) gel strip with plastic backing is applied to the surface of the horizontal sodium dodecyl sulfate (SDS)‐gel, is extremely simple and easy to perform. Due to the optimal flat‐to‐flat contact of the two gels, no size or edge trimming of the gels, no agarose overlays nor embedding gels are needed. The influence of IEF, performed with carrier ampholytes, Immobilines or immobilized pH gradients (IPG) with carrier ampholytes, on 2‐D maps is demonstrated. More protein has migrated into the IPG gel with carrier ampholytes, displaying an increased number of spots.
The two‐dimensional pattern of cellular proteins from mitogen stimulated human peripheral blood lymphocytes
Authors: Waldinger Dorothea , Braun Andreas , Cleve Hartwig
Published: May 2005

Human peripheral blood lymphocytes were isolated from healthy donors and cultivated in the absence and presence of phytohemagglutinin (PHA) or pokeweed mitogen (PWM). Cell lysates of unlabelled lymphocytes or following stimulation of [H]leucine metabolically labelled lymphocytes were subjected to two‐dimensional electrophoresis. Differences in the protein patterns are described which are related to the method applied for visualization of the proteins (silver staining or fluorography), to the culture conditions (unstimulated, PHA or PWM stimulated lymphocytes) and to individual variations. Comparative analysis of the resulting patterns on silver stained gels and fluorograms revealed three qualitative and several quantitative differences. Comparing the two‐dimensional patterns of unstimulated and mitogenstimulated lymphocytes with silver staining, we recognized the quantitative increase of 6 protein spots after stimulation. Whereas protein patterns of PHA and PWM stimulated cells showed only minor quantitative differences when compared in silver stained gels, one qualitative and two quantitative differences between the two mitogens could be observed in fluorograms. Individual variability was generally low: within the approximately 30 unrelated individuals examined, thus far, the presence of two polymorphic proteins was demonstrated.
An improved procedure for polypeptide analysis of radiolabeled antigens resolved by crossed immunoelectrophoresis and its application to the study of inner and outer membranes of Escherichia coli
Authors: Owen Peter
Published: May 2005

An improved procedure is described for polypeptide analysis of radiolabeled antigens resolved by crossed immunoelectrophoresis (CIE). The method involves detection of immunoprecipitates by autoradiography of CIE gels dried onto filter paper. This modification allows selected segments of immunoprecipitate arcs to be excised with a high degree of precision. Radiolabeled antigens are extracted from excised precipitates by incubation at 60 °C in Laemmli sample buffer, and polypeptides are visualized by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis performed in conjunction with autoradiography or fluorography. Protein antigens of the bacterial outer membrane are shown to retain (in part) their properties of heat‐modifiability following drying and extraction, thus facilitating their identification. The procedure is applied to the analysis of fourteen membrane‐associated antigens of Escherichia coli and results in the identification of the ompF/C and the ompA gene products, and the resolution of two novel heterooligomeric outer membrane protein antigens. The polypeptide composition of four previously uncharacterized inner membrane antigens is also established. In addition, six antigens which had been characterized by other unrelated methods as common protein antigen, β‐galactosidase, adenosine‐5′‐triphosphatase, dihydrolipoyl dehydrogenase, D‐lactate dehydrogenase and the lpp gene product (the Braun lipoprotein) are shown to possess polypeptide profiles which confirm the initial identification. Evidence is also presented to support the thesis that the bound form of the lpp gene product can associate with other proteins of the outer membrane.
A practicallaboratory method for cell electrophoresis in an agarose sol
Authors: Gilman Robert L.
Published: May 2005

The method presented in this article was developed specifically to be used for the electrophoretic separation of biological cells. This method uses low cost materials and ordinary clinical laboratory electrophoresis equipment. Results are reasonably fast; time of migration was 20 min using a constant current setting of 6 mA for a single 3 × 4 inch gel tray. The use of SeaPlaque agarose sol at 40 °C allows the cells to migrate freely, while the solidification of the agarose at the end of the migration period fixes the cells in place on the plate. Samples can be taken from any position on the plate while the sol is still liquid. With this method, differences in rate of migration of erythrocytes from selected species of mammals were demonstrated. The method was developed using erythorcytes for samples and without staining of gels or prestaining of samples. Sample sizes of 2000 to 4000 cells gave easily visible bands. Band of cells from some species migrated as much as 40 mm. Separations were done in polystryrene gel tryays. This technique will be tested in the near future for the separation of the various types of blood cells and for separating other cell mixtures, e. g., bacteria.
Analytical control of the production of herbicides and growth regulators glyphosate and glyphosine by capillary isotachophoresis
Authors: Křivánková Ludmila , Boček Petr
Published: May 2005

A method is described for direct analysis of the reaction mixtures of the production of the herbicide glyphosate and the growth regulator glyphosine by capillary isotachophoresis providing simultaneous determination of the products N‐phosphonomethyl‐glycine and N.N‐bis(phosphonomethyl)glycine as well as of all important reaction components ‐ sulfuric, formic, phosphorous, phosphoric, hydroxyacetic, nitrilotriacetic, iminodiacetic and N‐phosphonomethyliminodiacetic acids and glycine. The method is suitable for the analyses both of the final products and of mother liquors, where the maximum ratio of the concentrations ranges within 1:100. The time of analysis is dependent on the sample composition and ranges within 5–20 min. relative standard deviation of a single determination being less than 4 rel. %.
Utilization of highly repeated DNA sequences as a criterium of complete digestion on agarose gels: Example of human DNAs restricted by various endonucleases
Authors: Rahuel CÉCile , Dandieu Sylvie , Lucotte GéRard
Published: May 2005

Comparison between highly repeated DNA fragments of eucaryotic DNA digested by restriction endonucleases and the corresponding pattern of a completely digested DNA can be used as a criterium of complete digestion before the Southern blotting procedure. Results are presented for highly repeated fragments of human DNA, partially and completely restricted by six different endonucleases. Direct visualization of these fragments was obtained by ethidium bromide staining after agarose gel electrophoresis. Fragments corresponding to a complete DNA digestion were retained and can be used as reference.
Alpha‐amylase inhibitor in cereals: Comparison of the protein in different rye, wheat and triticale seeds by using immunoblotting
Authors: Sadowski Jan , Macgregor Alexander , Daussant Jean
Published: May 2005

Several isoforms of the barley α‐amylase inhibitor were detected in the purified protein fraction and seed extract after isoelectric focusing. Polymorphism not detected by protein staining was revealed by immunoblotting using an antiserum against barley α‐amylase inhibitor. The antiserum, previously described as giving cross reactions with the inhibitors of rye and wheat, was used for comparing the electrophoretic patterns of the protein in several lines of rye, in several varieties of wheat and in different triticales. The antiserum was used for immunoblotting after isoelectric focusing at pH 3.5–10. The inhibitor was found in all samples but the degree of polymorphism was low. The same pattern was found for all lines of rye investigated. This pattern differed from the pattern found for wheat, but again, all cultivars of wheat investigated displayed similar patterns. Structural genes for the α‐amylase inhibitors were identified in the A and R genomes. Identical genes may exist on the B and D genomes. The wheat and rye structural genes for α‐amylase inhibitor were found to be expressed in the octoploid, hexaploid and tetraploid triticales.
Ionophoretic technique in the study of mixed ligand complexes (M‐nitrilotriacetate‐threoninate systems)
Authors: Singh Satyendra , Bajpai Arun K. , Tripathi Sita R. , Yadava Kanhaiya L.
Published: May 2005

A novel ionophoretic technique for the study of complexes especially mixed ones has been described in the communications from this laboratory. Here with the help of this technique the stability constants of the complexes M‐nitrilotriacetate‐threoninates have been found to be 5.24, 5.01, 3.54 and 3.21, respectively.
Mitochondrial DNA in natural populations: An improved routine for the screening of genetic variation based on sensitive silver staining
Authors: Tegelström Håkan
Published: May 2005

When screening natural populations for genetic variation in mitochondrial (mt) DNA it is necessary to screen as many individuals as possible for as large a number of base pairs as possible. This report describes a combination of laboratory techniques – isolation of mtDNA by either density ultracentrifugation or a phenol extraction procedure and DNA fragment visualization by a sensitive silver staining technique. These routines remove the major restriction of tissue sample size and number of individuals that can be screened and allows the investigation of at least 1000 base pairs from 1 g of tissue or 3–400 base pairs from 100 mg of frozen tissue.
Separation of serum cholinesterase molecular forms from three rodent species by gradient polyacrylamide gel electrophoresis
Authors: Bisso Guillermo M.
Published: May 2005

Gradient (5–30 %) polyacrylamide gel electrophoresis (PAGE) was used for the analysis of serum cholinesterases from the spiny mouse, the mouse and the rat. Enzymatic staining of slab gels was performed using acetylthiocholine as substrate. Eserine, 1.5‐bis(4‐allyldimethylammoniumphenyl)‐pentane‐3‐one dibromide (BW284C51) and tetraisopropylpyrophosphoramide (iso‐OMPA) were used as selective specific inhibitors of total, true and pseudo‐cholinesterase, respectively. Five main molecular forms were detected in the three species as sharp, well separated bands. All bands were inhibited completely by eserine while the effects of BW 284C51 and iso‐OMPA varied in the three species. This result suggests a common qualitative pattern for the three rodents with a different distribution of true and pseudo‐cholinesterase molecular forms for each species. The good resolution shown by the reported densitometric scan recordings confirms the advantages of gradient over homogeneous PAGE in the study of serum cholinesterases.
FXIIIA polymorphism in a Japanese population: Occurrence of FXIIIA*4 allele
Authors: Suzuki Koichi , Matsui Kiyoshi , Matsumoto Hideo
Published: May 2005

The FXIIIA polymorphism was studied in a Japanese population using agarose gel isoelectric focusing and agarose gel electrophoresis, combined with immunoblotting onto nitrocellulose membranes. The correspondence of patterns between the two methods was confirmed by two‐dimensional electrophoresis. A rare phenotype, FXIIIA 4–1, was detected in one individual besides the three common phenotypes FXIIIA 1, 2–1, and 2. This rare phenotype was identified with the originally reported FXIIIA 4–1 in Melanesians. Our results indicate the possibility that the FXIIIA*4 allele may be distributed widely in world populations.
Fast electrophoresis in superposed agarose minigels for DNA topoisomerase assays
Authors: Hamelin Claude , Yelle Jocelyn , Dion Michel
Published: May 2005

Superposed miniature agarose slab gels and relatively small plasmids were used to evaluate the DNA topoisomerase I activity associated with the chromatin of normal human fibroblasts at three different in vitro population doubling levels. Results obtained in a short period of time with a single miniature electrophoresis unit confirm an increase in DNA‐relaxing enzyme activity with cell age.
Alkali enhancement of protein staining on nitrocellulose
Authors: Sutherland Mark W. , Skerritt John H.
Published: May 2005

Brief alkali treatment of nitocellulose‐bound protein markedly enhanced subsequent staining of protein with India ink or colloidal gold but not Amido Black. Following alkali treatment, India ink staining approaches colloidal gold staining in sensitivity and exceeds the latter method in clarity and economy. Furthermore, apparent protein‐to‐protein variation is reduced, allowing the method to be used for sensitive staining of protein transfers. Simple, high sensitivity staining of nitrocellulose‐bound protein using India ink can be achieved after alkali treatment in less than 2 hours.
Examination of isoelectric focusing and electrophoretic methods for resolving acidic proteins
Authors: Green Eric D. , Knecht David A. , Dimond Randall L.
Published: May 2005

Highly acidic proteins are difficult to study using conventional electrofocusing and electrophoretic techniques. For example, the lysosomal enzymes from the cellular slime mold Dictyostelium discoideum have isoelectric points (pI) around 3–4 due to extensive phosphorylation and sulfation. To assess differences in anionic modifications among these lysosomal enzymes, we required methods that resolved acidic proteins on the basis of their net charge. The separation procedures needed to be rapid and convenient, allowing parallel analysis of a large number of samples. Using these criteria, we developed two methods, isoelectric focusing in horizontal agarose gels and native electrophoresis in vertical polyacrylamide gels, which are more useful than other available techniques. Since the mechanism by which each system separates proteins is different, both methods give important information about acidic proteins.
Epigenetic modification of murine Cu,Zn‐superoxide dismutase
Authors: Shibata Futoshi , Ogita Zen‐Ichi
Published: May 2005

Murine Cu, Zn‐superoxide dismutase (SOD) from erythrocytes has three isozymes, SOD‐1A (pI 6.4), B (pI 5.7) and C (pI 5.0). It has been suggested that SOD‐1A is formed epigenetically by asymmetric modification of SOD‐1B where as SOD‐1C is produced by treatment of superoxide dismutase with hydrogen peroxide. On two‐dimensional gel electrophoretic analysis, each isozyme shows two forms, easily permuted into each other, and hence supposed to be in equilibrium. The zymogram patterns of murine liver, kidney, heart and brain are the same as those in erythrocytes although SOD activity levels are different in these tissues.
Isotachophoretic analysis of the pH dependent carrier properties of bovine serum albumin for rifamycin AF/013
Authors: Csordas Adam , Oefner Peter Josef , Bartsch Georg , Grunicke Hans , Lancini Giancarlo
Published: May 2005

The binding sites of bovine serum albumin for rifamycin AF/013 were examined by means of analytical capillary isotachophoresis. At pH 7.5–7.9, there was only one binding site on non‐defatted bovine serum albumin, while under the same conditions up to 20 binding sites could be detected on fatty acid‐free albumin. In both cases the interaction became nonspecific above pH 8.0, resulting in a large number of rifamycin AF/013 molecules bound to albumin. Co‐binding with oleic acid in vitro resulted in competition with all but the first high‐affinity binding site for rifamycin AF/013, corroborating the findings made with non‐defatted albumin. Considering albumin as a model, the results might explain the conflicting reports about the number of binding sites of DNA‐dependent RNA polymerases for rifamy cin AF/013. Within a sharp pH‐limit the highly specific binding becomes a nonspecific one. Moreover, the results suggest that the carrier function of bovine serum albumin might drastically change in vivo, depending on the pH of physiological fluids.
Subcellular localization of some wheat polypeptides revealed by two‐dimensional electrophoresis
Authors: Granier Fabienne , Thiellement Hervé , Ambard‐Bretteville Françoise , Rémy René
Published: May 2005

Cell fractionation experiments were undertaken in order to assign some of the spots revealed by two‐dimensional gel electrophoresis of seven‐day‐old wheat etiolated seedlings to plastids or mitochondria. Among the 766 spots of these two‐dimensional gels, 251 have been identified: 139 in etioplastic stroma, 139 in chloroplastic stroma, 59 in mitochondria and 23 in chloroplastic membranes. Among them, 83 are common to etioplastic and chloroplastic stroma fractions but only 11 spots are common to mitochondria and plastids sub‐fractions.
How to estimate the position of the mistranslated proteins on a two‐dimensional gel electrophoregram
Authors: Laughrea Michael , Filion Anne‐Marie , Boulet Louise
Published: May 2005

A method for the estimation of amino acid misincorporation into ribosomal proteins is presented. The method is based on the observation that the horizontal mobility of the 52 E. coli ribosomal proteins is proportional to Q/N, where Q is the calculated charge of the protein and N designates the number of residues in the protein. Thus the mobility change caused by a change in charge, such as the replacement of arginine by cysteine, can be predicted and the gel fractions used for scintillation counting cut accordingly. The gel system used in this paper was not the O'Farrell system but a ribosomal system whose first dimension was run at pH 5 in the presence of 8 M urea.
Application of two dimensional electrophoresis to characterise hormonally sensitive proteins in the normal and abnormal uterus
Authors: Guest Julian F. , Elder Murdoch G. , White John O.
Published: May 2005

Progesterone therapy of androgenized rats results in the elimination of histological abnormalities of the uterus associated with constant unopposed oestrogenic stimulation. Subcellular fractions prepared from uteri of normal, androgenized and progesterone treated animals have been analysed by two‐dimensional gel electrophoresis. In all fractions, protein changes were observed as a result of androgenization and subsequent progesterone therapy. The identity of some of these proteins has been assigned following peptide mapping. Following progesterone therapy, the relative proportion of cytosolic tubulins, tropomyosins and D‐glucose‐6‐phosphate dehydrogenase were increased. In the abnormal uterus, a protein with similarity to the B subunit of creatine kinase was present in the mitochondrial fraction but could not be detected in the cytosol. Subsequent to progesterone therapy, this protein was no longer detectable in the mitochondrial fraction whilst two molecular forms of the B subunit of creatine kinase appeared in the cytosol. These hormonally‐sensitive changes in both structural and enzymatic proteins, which are ubiquitous to most cell types, are discussed in relation to morphological differentiation of the uterus and the molecular mechanisms underlying cell growth.
Pollen allergen purification by three successive preparative electrophoretic steps
Authors: Demeulemester Claude , Peltre Gabriel , Panheleux Dominique , David Bernard
Published: May 2005

Water soluble allergens of grass pollen (Dactylis glomerata) were purified by two successive steps of preparative isoelectric focusing in a flat bed of granulated gel and controlled by analytical isoelectric focusing in agarose gels. In order to separate components cofocusing in the same pH zone but having different electrophoretic mobilities, preparative zone electrophoresis in granulated gels was performed. The homogeneity of the fractions recovered was tested by isoelectric focusing and zone electrophoresis, followed by radioimmunodetection of the allergenic molecules on nitrocellulose prints. The major allergen of Dactylis glomerata pollen was purified with a yield of 7.5 mg from 10 g of pollen grains.
Degradation kinetics of an alkaline Immobiline in the frozen state
Authors: Astrua‐Testori Silvia , Pernelle Jean‐Jacques , Wahrmann Juan P. , Righetti Pier Giorgio
Published: May 2005

The degradation kinetics of Immobiline pK 9.3 (0.2 m in H2O) have been studied over a period of 2‐years in the frozen state. The apparent hydrolysis rate (derived from the shift of myosin spots in a two‐dimensional map) is 20% per year (or 10% if incorporation of acrylic acid, an hydrolysis product, is accounted for). Such reactions in frozen solutions are not uncommon (cf. the spontaneous mutarotation of glucose, Kiovsky, T. E. and Pincock, R. E., J. Amer. Chem. Soc. 1966, 88, 4704–4710).
Two‐dimensional analysis of membrane proteins with isoelectric focusing in immobilized pH gradients in the first dimension
Authors: Gianazza Elisabetta , Caccia Paolo , Quaglia Laura , Righetti Pier Giorgio , Rimpiläinen Marja A. , Forsén Raili J.
Published: May 2005

Two‐dimensional electrophoresis with isoelectric focusing in immobilized pH gradients in the first dimension was applied to the fractionation of hydrophobic proteins from the plasma membrane of a Streptococcus strain. The detergents incorporated into the first‐dimensional gel slab, especially the zwitterionic ones of the sulfobetaine series, interfere with protein transfer from the first to second dimension by formation of mixed micelles with sodium dodecyl sulfate molecules. In order to reduce their concentration an elution protocol was devised including: (i) fixation for 1 h in 50 % methanol ‐ 12 % acetic acid; (ii) washing with distilled water for 30 min; (iii) equilibration in concentrated Tris buffer; (iv) denaturation in 5 % sodium dodecyl sulfate ‐ 2 % 2‐mercaptoethanol. When counting the number of resolved spots in the protein patterns after two‐dimensional electrophoresis, the relative solubilizing efficiency of different detergents scored as follows: Nonidet P‐40 (NP‐40) > (3‐[3‐cholamidopropyl)dimethylammonio]‐1‐propananesulfonate (CHAPS) > N‐dodecyl‐N, N‐dimethylammonio‐3‐propanesulfonate (sulfobetaine SB 12).
Determination of relative amounts of silver stained proteins on two‐dimensional gels using internal calibrator
Authors: Stastny Jaroslav J. , Fosslien Egil
Published: May 2005

Proteins separated by isoelectric focusing of 20 samples and polyacrylamide gel electrophoresis of 10 samples in the same batch were stained with silver and were scanned with a computerized laser beam densitometer to obtain integrations values. A linear relationship was found between densitometric integration values (staining intensities) of proteins and their loads up to 600 ng (actin, carbonic anhydrase) or 1000 ng (albumin, creatine kinase), respectively. The same amount (250 ng) of applied albumin, actin, carbonic anhydrase and creatine kinase resulted in differences in staining intensities of these proteins; staining intensities of albumin and creatine kinase were similar, however: both were 1.8 X higher than the staining intensity of carbonic anhydrase and 2.3 X higher than of actin. Six human aortic intima proteins (ML‐1, ML‐2, P27, −14, P27,−18, P27,−19.5, P110) were selected for quantitative studies of the relationship between loads of intimal extract and the integration values of these proteins. Linear responses were found for these proteins in loads from 5 μL to 20 μL of intimal extract, corresponding approximately from 15 μg to 60 μg of total proteins applied to the focusing gel. When carbonic anhydrase was included with each sample, as an internal absorbance calibrator, we could express integration values of other proteins in relative amount in ng of carbonic anhydrase and thus compensate for variation in fixation, developing and staining conditions from batch to batch.