bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isotachophoretic focusing on thin gel slab: A new and powerful electrophoretic method of protein analysis
Authors: Charlionet Roland , Bringard Aline , Davrinche Christian , Fontaine Marc 
Published: May 2005

AbstractThe theoretical principle and experimental verification of a new separation method for proteins in thin gel slabs called “isotachophoretic focusing” are presented. Isotachophoretic focusing is basically isotachophoresis with two new features: (i) By using adequate spacers a continuous mobility spectrum is generated. Under isotachophoresis conditions, these spacers give rise to a linear electric field gradient in which proteins can be separated in form of focusing bands. The synthesis and isolation of suitable spacers for isotachophoretic focusing are described. (ii) The stack is immobilized or, at least, its migration is retarded by exploiting the counterflow of electroendosmosis resulting from the action of the electric field on charged groups linked to the gel. Immobilization of the stack allows its formation to equilibrium and the protein separation process to continue as long as necessary. Details are given concerning the experimental set‐up and factors involved in the optimization of the technique. The separations obtained by isotachophoretic focusing are compared with those obtained by classical electrophoresis.
Transfer of proteins from plastic‐backed isoelectric focusing gels to nitrocellulose paper
Authors: Albaugh George P. , Chandra G. Ram , Bottino Paul J. 
Published: May 2005

AbstractProteins were transferred to nitrocellulose paper from plastic‐backed isoelectric focusing gels. The technique involves a two‐step blotting procedure. In the first step, simple press blotting was used to establish adhesion between the gel and overlaying nitrocellulose paper. This step facilitated the removal of the plastic backing. In the second step, electroblotting produced a pair of bi‐directional replica blots. Incubation of the gel‐nitrocellulose paper sandwich in buffers containing T ween 20 permitted the intact release of the gel from the blots. The resulting blots were successfully stained with India ink or probed with antiserum.
Enzyme polymorphism in bacteria: Study of molecular relatedness by combined isoelectric focusing‐electrophoresis
Authors: Picard Bertrand , Krishnamoorthy Rajagopal , Goullet Philippe 
Published: May 2005

AbstractThe molecular relationships between electrophoretic variants of different bacteria, defined in two‐dimensional electrophoretic profiles of acid phosphatase, esterases, glutamate dehydrogenase and malate dehydrogenases, were analyzed by combined isoelectric focusing‐electrophoresis. The mobility curves of enzymes confirmed the monomorphism of variants showing identical isoelectric point (pI) and relative electrophoretic mobility (MF) values and refined the polymorphism analysis of variants showing distinct pI and/or MF values.
Electrophoretic variability in human head hair: Polyacrylamide gel electrophoresis of hair proteins in the presence of sodium dodecyl sulfate and urea
Authors: Gerhard Michael 
Published: May 2005

AbstractA fast and simple procedure for routine typing of human head hair is described. Hair proteins, extracted in a solution containing sodium dodecyl sulfate, urea and dithiothreitol were separated electrophoretically in the presence of 6 M urea. On examination of hair from 445 different individuals, eight characteristic polypeptide patterns (phenotypes) could be distinguished and were arbitrarily named K1 to K8. Three of them, K1, K2 and K3 occur frequently, especially K1, which is found in approximately 79% of the individuals investigated. The eight phenotypes are characterized by the different number and patterns of major polypeptide bands in the range of 45 to 60 kDa. Based on the high reproducibility it can be assumed that a given phenotype is specific for the head hair of an individual. Electrophoretic keratin typing is a promising tool for hair analysis in genetic and forensic investigations.
A comparison of some adjustment techniques for use with quantitative spot data from two‐dimensional gels
Authors: Kuick Rork D. , Hanash Samir M. , Chu Ernest H. Y. , Strahler John R. 
Published: May 2005

AbstractQuantitative analysis of polypeptide differences among sets of identically prepared two‐dimensional polyacrylamide gels is facilitated by the use of mathematical transformations of the spot integrated intensity data for each gel in order to make comparisons. Such adjustments are needed because technical factors cause the stained polypeptide spots displayed on a particular gel to simultaneously appear darker or lighter than on another gel. We have compared three different adjustment schemes by applying each to the same set of data and testing to determine which gives smaller coefficients of variation for a large battery of spots. The adjustment that gave the best results is one based on regressing the spot integrated intensities of a standard gel versus each “target” gel and using the fitted line to transform the spot integrated intensities on the target gel. This adjustment is intuitive, easy to compute, and performed well regardless of whether the spots with large variability were included in the data or not.
Polystyrene latex particles as size standards in quantitative agarose gel electrophoresis: Application to three plant viruses
Authors: Gombocz Erich , Tietz Dietmar , Hurtt Suzanne S. , Chrambach Andreas 
Published: May 2005

AbstractNegatively charged polystyrene latex particles with electron microscopically determined radii of 22.4, 26.9, 27.6, 43.5, 44.2 and 59.4 nm (nominal radii provided by the manufacturer of 28.5 to 75 nm) were found to be applicable as size standards for the quantitative agarose gel electrophoresis of viruses and cellular particles in that size range. In electrophoresis at pH 6.5, 0.03 M ionic strength in presence of 10 mM 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate (CHAPS), the polystyrene particles exhibit non‐linear, reproducibly convex‐sigmoidal Ferguson plots. This curve shape is compatible with a model specifying a dependence of gel fiber dimensions and of particle size on the gel concentration. Based on a standardization of the agarose fiber by the polystyrene latex particles, computer simulation methods were used to determine the effective radii (without added Ca++) of turnip crinkle virus (TCV), hibiscus chlorotic ringspot virus (HCRSV) and pelargonium flowerbreak virus (PFBV) as 29.4, 24.2 and 22.1 nm, and their net charges as free mobilities, μo of 9.89, 10.87 and 16.20 · 10−5 cm2/s/V, respectively. Within the size range of the standards, a simplified procedure for determination of particle size from curved Ferguson plots appears applicable as a first approximation (Zwaan method). When electrophoresis was conducted in the presence of 5 mM Ca++, the effective particle radii of TCV, HCRSV and PFBV were reduced to 12.8, 20.3 and 18.3 nm, respectively. This compares with previous electron microscopic estimates of approximately 15 nm for TCV, HCRSV and PFBV. The observed increase of effective particle radii in the absence of Ca++ corroborates the particle swelling and/or shape change at pH's ≥ 5.5 in the absence of Ca++ which is known to occur with other plant viruses.
Protein precipitation induced by alkaline Immobilines for isoelectric focusing in immobilized pH gradients: Causes and remedies
Authors: Rabilloud Thierry , Gelfi Cecilia , Bossi Maria Luisa , Righetti Pier Giorgio 
Published: May 2005

AbstractThe mechanism by which three basic Immobilines (pK's 6.2, 7.0 and 9.3) precipitate proteins in solution seems to be due to the presence in these three monomers of variable amounts of oligomers, spontaneously formed during storage by polymerization. These polymerization products range in size from short oligomers (dimers and trimers) to polymers with a molecular mass more than 2000 Da. Oligomers are present in all batches of precipitating Immobilines in substantial amounts, usually at least 15–20%. Only the pK 8.5 Immobiline, in general unable to precipitate proteins from solution, has been found to contain low levèls of oligomers, typically less than 3%, possibly due to intrinsic stability against polymerization, or to its ability to spontaneously hydrolyze in water solutions. None of the batches of pK 6.2, 7.0 and 9.3 Immobilines, unable to precipitate proteins in solution, was found to contain polymers but exhibited a bimodal elution peak from a Bio‐Gel P‐2 sieve gel chromatography, probably corresponding to dimers and trimers. Upon mild polymerization (highly diluted persulfate in presence of oxygen) all these batches were found to precipitate proteins and to contain higher oligomers and polymers. By testing column eluates, it was found that the precipitation power begins at the level of polymers with 10–12 residues, suggesting that these relatively short chains already possess a strong cooperativity in their chemical behavior in solution. Work is in progress to find a suitable solution for completely inhibiting two of the most noxious reactions occurring to Immobilines: hydrolysis and polymerization. There still remains a third problem, which will require a longer‐term investigation: the intrinsic hydrophobicity of the four basic Immobilines. The basic Immobiline homopolymers present two unlike groups for interaction: ionic on one side, hydrophobic on the opposite end. Acidic Immobilines, even when reacted to form much larger polymers, are unable to precipitate ferritin from solution, suggesting that their strongly hydrophilic surface suppresses one of the two binding regions. In a similar manner, when batches of pK 7.0 Immobiline, able to precipitate ferritin, where rendered more hydrophilic by methylolation, their protein precipitation power was abolished, even, though the polymers had not been eliminated. While waiting for the synthesis of more ‘hydrophilic’ Immobilines, a partial remedy is to admix carrier ampholytes (CA) to Immobiline gels. The CA's act as shielding ions, minimizing hydrophobic interactions among proteins and the Immobiline matrix. Mixed CA‐IEF gels should never be prefocused, as the shielding action of CA is maximal in the unfocused state.
A micromethod for typing haptoglobin in bloodstains
Authors: Dimo‐Simonin Noëlle , Brandt‐Casadevall Conxita , Gujer Hans‐Rudolf 
Published: May 2005

AbstractA micromethod of discontinuous polyacrylamide gel electrophoresis is described for phenotyping human haptoglobin in bloodstains. Pretreatment of samples is not required. This procedure is easy, fast and can detect haptoglobin phenotypes in 0.02 μL blood in experimentally produced stains.
A tetrazolium method for peroxidase staining: Application to the antibody‐affinity blotting of α‐fetoprotein separated by lectin affinity electrophoresis
Authors: Taketa Kazuhisa 
Published: May 2005

AbstractA sensitive staining method of peroxidase labels on nitrocellulose membrane was developed with phenol as an enzyme substrate and NADH as a reducing agent for Nitro Blue tetrazolium to form formazan. The tetrazolium method for peroxidase staining, when applied to the antibody‐affinity blotting of α‐fetoprotein (AFP) after agarose gel electrophoresis, allowed us to detect as little as 3 ng/mL AFP with 4 μL of sample volume, covering a wider linear range of color production up to 200 ng/mL AFP as compared with the conventional diaminobenzidine stain. Accordingly, the proportion of intensities of AFP bands separated by lectin affinity electrophoresis could be determined without employing a calibration curve for each assay.
Subtyping of factor B by agarose gel electrophoresis
Authors: Suzuki Koichi , Harumoto Terue , Ito Shigenori , Matsumoto Hideo 
Published: May 2005

AbstractConventional agarose gel electrophoresis (AGE) in Tris/glycine/barbital buffer (TGVB) could classify the common allotype “F” of factor B (BF) into two kinds of subtypes (Fb1 and F). Analysis of mother‐child pairs in paternity cases confirmed inheritance of these suballeles in the same manner as other alleles at the BF locus. The gene frequencies were estimated at 0.0154 for BF*Fb 1 and 0.1492 for BF*F through the study of 325 unrelated individuals. After treatment of serum samples with zymosan, the polymorphic band of BFFb 1 appeared in the Ba fragment, migrating between those of BFF and BFS. A technique of immunoblotting could demonstrate the BF patterns after AGE. This study provides a simple method for the detection of the BF subtypes and their electrophoretic characterization.
High resolution two‐dimensional electrophoresis of the proteins and macromolecular constituents of beer and wine
Authors: Marshall Thomas , Williams Katherine M. 
Published: May 2005

AbstractThe proteins/macromolecular constituents of unconcentrated/unfractionated beer and wine (50–75 μL) have been analyzed by high resolution two‐dimensional electrophoresis and silver staining. The beer patterns are dominated by up to four horizontal strings of spots of Mr 34 000–43 000 and pI 5.0–7.0 which have been tentatively identified as proteins previously isolated in association with foam and haze formation. Reproducible qualitative and quantitative differences were detected in different commercial beers. Distinctive patterns were also obtained with the wines.
Adsorption, immunoadsorption and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of proteins using nitrocellulose membrane filters
Authors: Luescher Immanuel F. 
Published: May 2005

AbstractA rapid analytical procedure is described to perform sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of proteins specifically adsorbed on antibody‐coupled nitrocellulose membrane filters. Purified antibodies were adsorbed on nitrocellulose filters and were immobilized by treatment with dilute glutaraldehyde. Efficient adsorption of specific antigen occurred upon filtration of the antigen solution (e. g. cell supernatant or cell lysate) within a few minutes. Theim‐munoadsorbed proteins were released from the membrane filters in the presence of urea, Nonidet P‐40 detergent and electrophoresis sample buffer and were directly analyzed by SDS‐PAGE. All adsorptions and washing steps were performed by filtration utilizing a filtration manifold. This allowed the processing of a large number of samples in a short time. Furthermore, and in contrast to the Western blotting method, antigen‐antibody recognition preceeded gel electrophoresis, which greatly reduced potential failures in antigen recognition. Similarly, SDS‐PAGE of proteins non‐specifically adsorbed on nitrocellulose filters was also possible. Dilute protein solutions, like cell supernatants, were filtered through nitrocellulose membrane filters. Hereby proteins, but not low molecular weight compounds such as inorganic salts, amino acids or detergents, were rapidly and quantitatively adsorbed. The adsorbed proteins were released from the filters and subjected to SDS‐PAGE as in the previous method.
Differentiation between Haemophilus paraphrophilus, H. aphrophilus, H. influenzae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, P. haemolytica, and P. ureae by high resolution two‐dimensional protein electrophoresis
Authors: Olsen Ingar , Rosseland Sissel K. , Thorsrud Anne K. , Jellum Egil 
Published: May 2005

AbstractThe cellular proteins of Haemophilus paraphrophilus (ATCC 29242, ATCC 29241T, ATCC 29240, HK 477, HK 319, HK 159), H. aphrophilus (ATCC 33389T), H. influenzae (ATCC 31441), Actinobacillus actinomycetemcomitans (ATCC 33384T), Pasteurella multocida (NCTC 10322T), P. haemolytica (9380T), and P. ureae (NCTC 10219T) were analyzed by high resolution two‐dimensional protein electrophoresis and silver staining. The electrophoretic protein patterns of Haemophilus, Actinobacillus and Pasteurella were quite distinct. Also the patterns of P. multocida, P. haemolytica and P. ureae differed markedly. Except for H. paraphrophilus ATCC 29242, it was difficult to distinguish H. paraphrophilus from H. aphrophilus strains. Excluding ATCC 29242, H. paraphrophilus strains were quite similar. A. actinomycetemcomitans could easily be distinguished from all species tested, including the related H. aphrophilus, H. paraphrophilus, P. haemolytica, and P. ureae. High resolution two‐dimensional protein electrophoresis is likely to contribute to a more unifying concept of bacterial species. Among the organisms examined only H. aphrophilus and H. paraphrophilus seem to requires revision of their current taxonomic positions.
Effect of sialic acid on the electrophoretic mobility of mouse splenic lymphocytes
Authors: Shimizu Motomu , Iwaguchi Takao 
Published: May 2005

AbstractThe effect of sialic acid on the electrophoretic mobility (surface charge) of mouse splenic lymphocytes was investigated. On treatment of whole spleen and T cells with neuraminidase, their electrophoretic mobilities decreased to one‐half of the initial values. Calculation of the surface charge from the electrophoretic mobility of lymphocytes before and after treatment with the enzyme indicated that one‐half of the negative surface charge, which determines the electrophoretic mobility, was due to neuraminidase‐sensitive sialic acid in mouse lymphocytes. However, the calculated decrease in the surface charge did not coincide with the amount of sialic acid released from the cell membrane on the enzyme treatment of lymphocytes.
Identification of bovine fetal and adult serum/plasma proteins by two‐dimensional electrophoresis and immunochemical staining
Authors: Manabe Takashi , Takahashi Yuko , Okuyama Tsuneo 
Published: May 2005

AbstractBovine fetal and adult serum/plasma proteins were analyzed by two‐dimensional electrophoresis in the absence of denaturants. Bovine serum proteins were identified by electrophoretic transfer from gel slabs followed by immunochemical staining. Amine oxidase was identified by activity staining. Antisera against human plasma proteins were employed for the immunochemical identification, together with antisera to bovine proteins. Developmental changes of fetal serum proteins including α‐fetoprotein were visualized.
Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of human salivary proteins including pink‐violet components: Range, distribution and response to 2‐mercaptoethanol
Authors: Marshall Thomas , Williams Katherine M. 
Published: May 2005

AbstractMid‐morning specimens of unstimulated whole saliva were collected from 160 apparently healthy individuals and analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis following sample preparation with or without 2‐mercaptoethanol. Coomassie Brilliant Blue R‐250 staining revealed blue and pink‐violet salivary protein components. The latter, detected in approximately 60 % of the individuals, formed a limited range of patterns combining 2–8 of 11 diffuse zones of Mr 10 000–100 000. The pink‐violet components were unaffected by 2‐mercaptoethanol, urea or prior centrifugation of the sample and were not invariably present or absent in repeat samples from the same individual. The patterns of the blue staining components were more reproducible for the purpose of human individualisation but were affected by 2‐mercaptoethanol or prior centrifugation of the sample. The patterns of both the blue and pink‐violet components were not affected by sample storage at −70 °C. Only 5 μL of saliva were required for analysis giving excellent resolution over the Mr range 10 000–500 000.
Separation accuracy of free flow electrophoresis as proved by flow cytometry
Authors: Bauer Johann , Kachel Volker 
Published: May 2005

AbstractThe separation accuracy of the free flow electrophoresis ACE 710 device was proved by immunological methods. Several cell populations of human peripheral blood were purified using physical cell isolation methods such as countercurrent centrifugal elutriation and cell electrophoresis. The purified cell fractions were treated with fluoresceinisothiocyanate‐labelled antibodies. The percentages of cells in each fraction binding the antibodies of interest were determined by flow cytometry. The analyses revealed that with the help of free flow electrophoresis, given cell population of human peripheral blood can be highly enriched and that preenrichment of minor cell populations enhances the efficacy of a flow cytometer.
Effect of salt on the performance of immobilized pH gradient isoelectric focusing gels
Authors: Strahler John R. , Hanash Samir M. , Somerlot Luke , Bjellqvist Bengt , Görg Angelika 
Published: May 2005

AbstractThe effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding ∼ 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion‐containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1–2 mM Tris base, or at the anode for gels rehydrated with 1–2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris‐containing gels, or at the cathode, for acetate‐containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading. Salt concentrations in excess of ∼ 0.15 M in the sample result in electroosmotic removal of water from the sample before the proteins can enter the gel, with resultant precipitation of the sample at the application site. Local pH extremes can be created at the application point, which may produce delayed sample entry or protein precipitation.
Physical identification of a virus in a crude leaf extract by its ferguson plot in agarose gel electrophoresis
Authors: Orbán László , Chrambach Andreas 
Published: May 2005

AbstractCrude extracts of turnip crinkle virus upon agarose gel electrophoresis yield (i) virus patterns unperturbed by contaminants; (ii) plots of mobility vs. gel concentration (Ferguson plots) parallel with those of the purified virus. The parallelism suggests similarity in size and shape but a lower net charge for the crude virus. This result is obtained when gel electrophoresis is carried out either in a continuous buffer or in a discontinuous (moving boundary electrophoresis) buffer system. The latter mode has the substantial benefit of electrophoretic (auto‐)concentration of dilute virus sample prior to resolution. Thus, the Ferguson plot analysis in a discontinuous buffer system of turnip crinkle virus can be viewed as a model procedure for the physical identification of other viruses contained in dilute extracts, feasible even in the absence of a prior knowledge as to the nature of, or isolation of, the virus.