bluuebunny/crossref_metadata_2025_split huggingface dataset:
The purification and characterization of bovine salivary proteins by electrophoretic procedures
Authors: McLaren Robert D. , McIntosh James T. , Howe Gregory W. 
Published: May 2005

AbstractBovine salivary proteins have been shown to be correlated with bloat susceptibility in cattle. Due to the presence of the high molecular weight mucoprotein in whole saliva these proteins have been difficult to purify. This difficulty has now been overcome by using electrophoretic procedures. The purification protocol consisted of electrophoretic concentration of the saliva, separation of the proteins by polyacrylamide gel electrophoresis, excision of the protein bands followed by electroelution and concentration. Purified proteins were obtained from bands 4, 5, 6, 7, 8 and 9/10. The molecular weights of the intact proteins were between 40 300 and 112 400 on gel chromatography and the subunit molecular weights were between 15000 and 65 400. The isoelectric points were within the pH range of 3.6 to 5.1. Antisera were produced against protein bands 7 and 8 and these were used to determine antigenic relationships between proteins and as a means of quantifying the proteins by the Laurell rocket immunoassay.
Genetic studies of human apolipoproteins. II. A rapid one‐dimensional isoelectric focusing technique to characterize apolipoproteins A‐I, A‐II, A‐IV and C‐II of unfractionated plasma
Authors: Kamboh Mohammad I. , Ferrell Robert E. , Sepehrnia Bahman 
Published: May 2005

AbstractA simple and rapid one‐dimensional analytical isoelectric focusing method followed by immunoblotting is described which detects genetic and biochemical variation in apolipoproteins A‐I, A‐II, A‐IV and C‐II without prior ultracentrifugation and delipidation of plasma samples. A polipoproteins separated on an isoelectric focusing gel are sequentially transferred to two nitrocellulose membranes and pairs of apolipoproteins are identified on each membrane by immunological reactions. The specificity of the primary antibody and the sensitivity gained by use of a second, alkaline phosphatase conjugated anti‐IgG allows the detection of free apolipoproteins in serum or plasma. A pH gradient 4–6.5 is suitable to identify the four apoproteins tested from a single gel and there is little loss of isoprotein resolution in sequential blots. Only 2 μL of whole plasma is required and results on 50 samples can be obtained from a single horizontal gel within one day.
Double replica electroblotting by oscillating electrotransfer
Authors: Johansson Karl‐Erik 
Published: May 2005

AbstractProtein blotting has become an indispensable tool in biochemistry and in many blotting experiments it is desirable to obtain more than one replica from the gel, which would permit the use of different probes or staining of one membrane for protein and using the other(s) for probing. This has proved difficult. The different replicas, which can be produced by existing techniques, are far from identical. However, by using oscillating electrotransfer “double replica electroblotting”, two replicas can be obtained from one gel. The two replicas can be made almost identical by reversing the direction of the current in such a way that the efficient transfer time is increased with each period until the gel is almost completely depleted of proteins. The technique is useful for comparison of the reactions of different antisera with one or several antigen samples. Double replica electroblotting has been utilized for ordinary sodium dodecyl sulfate‐polyacrylamide gels, for two‐dimensional polyacrylamide gels and for thin gradient gels analyzed with the PhastSystem (Pharmacia). Double replica electroblotting can, in principle, be used with any blotting system where transfer from the gel to the new medium is achieved by electrophoresis, except in semi‐dry electroblotting with a discontinuous buffer system.
Visualization of molecular weight standards after electroblotting: Detection by means of corresponding antibodies
Authors: Bjerrum Ole J. , Hinnerfeldt Frank R. 
Published: May 2005

AbstractThe techniques available for detection of molecular weight standards after blotting are surveyed and evaluated. An identical immunodetection procedure for the antigens as well as for the molecular weight markers was evaluated by immunizing rabbits with the whole protein mixture of some commercially available calibration kits for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Antibodies against both high and low molecular weight proteins were successfully raised albeit one rabbit failed to generate antibodies against ovalbumin. For immunodetection of rabbit myosin it was necessary to supplement the immunogen with bovine myosin. Best results were obtained by using more than one animal immunized with kits of different origin. Using alkaline phosphatase conjugated secconary antibodies, as little as 0.6 and 1.2 μg of the total low and high molecular weight mixture, respectively, are necessary for the immunoenzymatic detection of all bands. In fused rocket immunoelectrophoresis such antibodies are also useful for determination of the elution position of the molecular weight markers in gel filtration experiments.
Characterization of autoantigens and autoantibodies by immunoblotting
Authors: Elkon Keith B. , Bonfa Eloisa , Li‐Chu Jia , Parnassa Andrew P. 
Published: May 2005

AbstractImmunoblotting has proven to be one of the most useful methods for detection of antibodies and partial characterization of proteins that are antigenic in the autoimmune diseases. The basic physicochemical characteristics (size and charge) of most of the major autoantigens have now been described. By combining various electrophoretic techniques with immunoblotting, a number of new applications have been reported. Two‐dimensional electrophoresis was used to fingerprint autoantigens and detect post‐translational modifications. Composite gel electrophoresis resolved ribonucleprotein complexes and was also used to analyze IgA, IgM autoantibodies and to determine the composition of cryoglobulins. Autoantibody heterogeneity was analyzed by isoelectric focusing. In some studies, the subclass, idiotype, or antigen binding specificity of autoantibodies could be determined by immunoblotting procedures. Coupled with improvements in antigen purity (synthetic peptides and recombinant proteins) and wider availability of monospecific antibodies, immunoblotting will continue to be a versatile tool in basic and clinical research in autoimmunity.
Factor B polymorphism in four Asian populations: Electrophoretic characterization of a subtype of “F” and of new variants in the Chinese
Authors: Suzuki Koichi , Charoenwongse Preeyachit , Lingao Augusto L. , Contractor Nafisa M. , Matsushita Sho , Sasazuki Takehiko , Matsumoto Hideo 
Published: May 2005

AbstractThe genetic polymorphism of factor B (BF) in four Asian populations was studied by immunofixation after agarose gel electrophoresis (AGE) in both veronal buffer (VB) and Tris/glycine/veronal buffer (TGVB). A suballele of the BF*F and three rare alleles were detected in a Chinese population. The subtype and the rare variants were further characterized by using isoelectric focusing in polyacrylamide gels (PAGIF) and by zymosan treatment of the serum samples. The subtype was identified as BFFb1, first defined in the Japanese and found to occur at a polymorphic frequency. One of the rare variants exhibited an intermediate mobility between BFF and BFS and seemed to be similar to BFF025. PAGIF, however, could clearly discriminate the two variants that had a similar mobility on AGE. The results obtained in this study suggest that conventional electrophoresis is not enough to characterize a new variant and to discriminate it from previously reported variants. The findings obtained on immunofixation after AGE/TGVB and in PAGIF must be added to those obtained by the standard method.
Electrophoretic variability in human hair: Comparative sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of body and head hair proteins
Authors: Gerhard Michael , Hermes Monika 
Published: May 2005

AbstractThe electrophoretic patterns of extracted proteins from human head hair were compared to those of proteins from other human hair species (pubic, breast, beard, arm, leg. axillary and eyebrow hair). The respective patterns (keratin phenotypes) were shown to be uniform throughout one individual's hair. Keratin typing could be performed successfully subsequent to ABH blood group determination by mixed cell agglutination with the same hair material.
Analysis by two‐dimensional gel electrophoresis of the polypeptide composition of pea mitochondria isolated from different tissues
Authors: Remy René , Ambard‐Bretteville Françoise , des Francs Catherine Colas 
Published: May 2005

AbstractThe polypeptide composition of purified mitochondria isolated from pea epicotyls was studied by one‐ and two‐dimensional electrophoresis. It was shown that two‐dimensional electrophoresis was necessary to clearly resolve polypeptides of molecuar weights ranging from 50 000 to 60 000. Mitochondria were fractionated into a soluble fraction corresponding to the matrix and an insoluble fraction containing membrane components. Spots characteristic of one or the other fraction were thus localized on the two‐dimensional gel map of whole mitochondria. In addition, a comparison was made of polypeptide composition in mitochondria isolated from etiolated and green leaves. It was shown that mitochondria from photosynthetic tissue differ from those on non‐photosynthetic tissue by quantitative and qualitative changes of their polypeptide composition. The most characteristic changes in green leaves are the loss of one polypeptide (Mr 35 000 and pI 6.3) and the quantitative increase of four other spots. These spots belong to the matrix compartment. Their apparent molecular weights are 97 000, 51 000, 43 000 and 15 000. Conversion of glycine to serine during the photorespiration process being characteristic of mitochondria from photosynthesizing tissue, we suggest that these spots could correspond to subunits of the glycine decarboxylase multienzyme complex.
Improved Nitro Blue Tetrazolium staining of creatine kinase isoenzymes following agarose gel electrophoresis or isoelectric focusing in polyacrylamide gels
Authors: Williams John , Marshall Thomas 
Published: May 2005

AbstractAn improved method is described for Nitro Blue Tetrazolium staining of creatine kinase isoenzymes following agarose gel electrophoresis or isoelectric focusing in polyacrylamide gels. The method allows the standard although transient fluorescent stain to be followed by Nitro Blue Tetrazolium staining to give a more permanent positive stain without background interference. It employs. 0.05 M para‐chloromercuribenzoate to block the adverse effects of sulfhydryl compounds (included in commercial creatine kinase‐reagents) upon Nitro Blue Tetrazolium staining.
Alpha‐1 acid glycoprotein analysis on immobilized pH gradients
Authors: Gianazza Elisabetta , Catto Nadia , Righetti Pier Giorgio , Bianchi‐Bosisio Adriana 
Published: May 2005

AbstractBy taking advantage of the insensitivity of immobilized pH gradients to high protein and salt loads, microheterogeneity and polymorphism of α1‐acid glycoprotein could be demonstrated on whole serum by conventional Coomassie Brilliant Blue G‐250 staining. Different running conditions were compared for optimal resolution.
Immobilized pH gradients: Effect of salts, added carrier ampholytes and voltage gradients on protein patterns
Authors: Righetti Pier Giorgio , Chiari Marcella , Gelfi Cecilia 
Published: May 2005

AbstractSalts formed from strong acids and bases (e. g. NaCl, Na2SO4, Na2HPO4), presentin a protein sample applied to an immobilized pH gradient (IPG) gel, induce protein modification (oxidation of iron moiety in hemoglobin) already at low levels (5 mM) and irreversible denaturation (precipitation) at higher levels (>50 mM). This effect is due to production of strongly alkaline cationic and strongly acidic anionic boundaries formed by the splitting of the salt's ion constituents, as the protein zone is not and can not be buffered by the surrounding gel until it physically migrates into the gel matrix. Substitution of “strong” salts in the sample zone with salts formed by weak acids and bases, e. g., Tris‐acetate, Tris‐glycinate, Good's buffers such as (N‐[2‐aceta‐mido]‐2‐iminodiacetic acid (ADA)), (2‐[(2‐amino‐2‐oxoethyl)‐amino] ethanesulfonic acid (ACES)), (3‐[N‐morpholino] propane sulfonic acid (MOPS)), essentially abolishes both phenomena, oxidation and irreversible denaturation. Suppression of “strong” salt's effects is also achieved by adding, to the sample zone, carrier ampholytes in amounts proportional to the salt present (e. g. by maintaining a salt: carrier ampholytes molar ratio of at least 1:1). This suppression is due to the strong buffering power of the added carrier ampholytes, able to counteract drastic pH changes in the two moving boundaries. A reduction of these deleterious effects of strong salts is also achieved when the IPG run is performed at low voltage for a prolonged time (4 h at 500 V instead of only 1 h at 500 V, before switching to high‐voltage settings). Guidelines are given for trouble‐free IPG operations.
Automatic classification of two‐dimensional gel electrophoresis pictures by heuristic clustering analysis: A step toward machine learning
Authors: Appel Ron , Hochstrasser Denis , Roch Christian , Funk Matthieu , Muller Alex F. , Pellegrini Christian 
Published: May 2005

AbstractThe interpretation of two‐dimensional gel electrophoresis (2‐DGE) profiles can be facilitated by artificial intelligence and machine learning programs. We have incorporated into our 2‐DGE computer analysis system (termed MELANIE‐Medical Electrophoresis Analysis Interactive Expert system) a program which automatically classifies 2‐DGE patterns using heuristic clustering analysis. This program is a step toward machine learning. In this publication, we describe the classification method and the preliminary results obtained with liver biopsy electrophoretograms. Heuristic clustering is also compared to other classification techniques.
High resolution two‐dimensional electrophoresis and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of cheese proteins after rapid solubilisation
Authors: Marshall Thomas , Williams Katherine M. 
Published: May 2005

AbstractThe proteins of cheese are rapidly solubilised by heating to 95 °C in buffered 2 % sodium dodecyl sulfate, 5 % 2‐mercaptoethanol. Electrophoretic analysis of the solubilised proteins by either one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis or high resolution two‐dimensional electrophoresis yields reproducible patterns characteristic of an individual cheese and its extent of ripening. The patterns reveal (i) the residual amounts of milk casein and whey proteins, and (ii) the appearance of casein degradation products, including pink‐violet components as detected by Coomassie Blue staining.
Demonstration and elimination of artifacts associated with gold staining of proteins on nitrocellulose membranes after separation by sodium dodecyl sulphatepolyacrylamide gel electrophoresis
Authors: Bath M. L. 
Published: May 2005

AbstractArtifacts associated with gold staining of polypeptides separated by sodium dodecyl sulphate – polyacrylamide gel electrophoresis and transferred to nitrocellulose are eliminated by alkylation of reduced samples or by deletion of the reducing agent.
PI Mtoyoura: A new PI M subtype found by separator and hybrid isoelectric focusing
Authors: Yuasa Isao , Suenaga Kazuyuki , Umetsu Kazuo , Inagaki Osamu , Ikebuchi Jun , Ito Keiichi , Okada Kichiro 
Published: May 2005

AbstractIn the course of a population study of α1‐antitrypsin polymorphism by separator isoelectric focusing, a variant phenotype having a somewhat narrower spacing than PI M1M3 was observed in a Japanese blood donor. Family studies by hybrid isoelectric focusing in a carrier ampholyte‐supplemented immobilized pH gradient from 4.35–4.65 revealed that the products of the responsible gene, PI*Mtoyoura, were extremely close but slightly cathodal to those of PI*M1. The difference in isoelectric point between them corresponded to the resolving limit of isoelectric focusing. For this reason, although the propositus' father was deduced to have the genotype PI*M1/PI*Mtoyoura, the products of these two genes failed to form a double band pattern. Thus, the gene frequency for PI*Mtoyoura was unknown. These findings, however, indicate that a further microheterogeneity in the PI M subtype exists at least in the Japanese.
Somatic mutation studies in human lymphoid cells: The detectability of quantitative genetic variants in two‐dimensional gels
Authors: Kuick Rork D. , Hanash Samir M. , Strahler John R. 
Published: May 2005

AbstractThe feasibility of detecting quantitative genetic variants based on a decrease in the integrated intensity of polypeptide spots in two‐dimensional polyacrylamide gels of human lymphoblastoid cell clones was investigated. A battery of 65 spots on 115 gels was studied to determine the distribution of quantitative measures for spots where no mutation had occurred. The corresponding distribution for spots which have decreased integrated intensity as a result of a mutation at one of two alleles coding for the spot was investigated by quantitating spots for which mutations were known to have occurred. These two distributions allowed the estimation of the rates of false positive and false negative errors for any particular strategy aimed at detecting null mutations, and thus provides a basis for the design of efficient strategies. Our silver stained gels have sufficient reproducibility of spot integrated intensities so that, for situations in which the mutation rate is relatively high, it is practical to monitor a sub‐set of spots for null variants using the same digitized images as are used to detect structural variants.
Isoelectric focusing of group specific component in semen and vaginal stains on ultrathin immobilized pH gradient gels
Authors: Pflug Werner , Eberspächer Brigitte , Bäßler Gerhard 
Published: May 2005

AbstractSubtyping of the group specific component in secretions of semen and vaginal fluids is impossible with conventional detection systems. By means of a highly sensitive alkaline‐phosphatase secondary antibody system the group specific component can reliably be‐detected in semen stains. Results with vaginal swabs were inconsistent and in saliva stains Gc activity could not be detected.
Heterogeneity in copper and glycan content of ceruloplasmin in human serum differs in health and disease
Authors: Hansen John‐Erik S. , Heegaard Peter M. H. , Jensen Søren P. , Nørgaard‐Pedersen Bent , Bøg‐Hansen Thorkild C. 
Published: May 2005

AbstractCrossed immunoelectrophoresis of human serum revealed two heterogeneity types of ceruloplasmin with different electrophoretic migration. The two types both consisted of peptides with Mr 150 000, 100 000 and 45 000, which were interpreted as native ceruloplasmin and two hydrolytic fragments. The two types were different in copper content, and one type could reversibly be changed into the other. The glycan microheterogeneity of ceruloplasmin was analyzed by crossed affinommunoelectrophoresis with free Lens culinaris agglutinin (LCA) and wheat germ agglutinin (WGA). A third of the ceruloplasmin molecules, both high and low copper type, bound to LCA and two thirds to WGA. The heterogeneity and the microheterogeneity of ceruloplasmin in two groups of patient sera were compared to sera from healthy individuals. The ceruloplasmin type with respect to copper content was a much better factor than either glycan microheterogeneity or total serum concentration in discriminating between the three groups.
A double‐label two‐dimensional procedure for the analysis of membrane proteins
Authors: Wheeler Thomas T. , Jordan T. William , Ford Henry C. 
Published: May 2005

AbstractA previously described double‐label two‐dimensional electrophoresis procedure (Wheeler et al., Anal. Biochem. 1986 159, 1–7) for the analysis of differences between two complex mixtures of soluble proteins has been modified to allow analysis of proteins requiring detergent for aqueous solubility. The samples are first disrupted by sonication and the insoluble proteins concentrated by high‐speed centrifugation. The proteins are then solubilized with sodium dodecyl sulfate and further concentrated in a centrifugal concentrator to achieve protein mixtures suitable for labeling with 14C and 3H by reductive methylation and subsequent two‐dimensional electrophoresis. The sample concentration step is quick, minimizes the concentration of sodium dodecyl sulfate in the final sample, and avoids the potential difficulties associated with lyophilization or precipitation. The modified procedure was applied to the analysis of erythrocyte membranes, platelets and isolated placental microvilli. The high resolving power of two‐dimensional gel electrophoresis is retained and the procedure is sensitive because the conditions of labeling allow substantial incorporation of radioactivity into protein despite the presence of detergent.
Comparative study of electrophoretic patterns of latex proteins from clones of Hevea brasiliensis
Authors: Arreguín Barbarín , Lara Paulina , Rodríguez Roberto 
Published: May 2005

AbstractLatex serum proteins from Hevea brasiliensis were studied by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Proteins from whole serum and fractions isolated by gel chromatography on Ultrogel AcA 44 were analyzed. No qualitative clonal differences were found in the protein patterns of whole latex or in the fractions but laser densitometry revealed reliable quantitative differences in protein composition. Reproducible mobilities and molecular weights of selected bands were obtained both within single gels as well as in different gels, analyzing severallots of latex received at various times from a Hevea experimental field station. The clones compared were IAN 710, GV 31, GV 42; the first one had the highest rubber yields.
Optimized oligonucleotide probes for DNA fingerprinting
Authors: Schäfer Renate , Zischler Hans , Birsner Uli , Becker Andrea , Epplen Jörg T. 
Published: May 2005

AbstractThe three different simple repetitive oligonucleotide probes (CT)8, (CAC)5 and (TCC)5 were hybridized to a panel of human DNAs which had been digested with the restriction endonucleases Alu I, Hinf I and Mbo I. The resulting DNA fingerprints were analyzed and different parameters calculated, such as the maximal mean allele frequency and the average number of polymorphic bands per individual. The highest number of bands was obtained after hybridization of Hinf I digested DNA with (CAC)5. The probability of finding the same band pattern as in individual A in individual B is 2 × 10−8. The DNAs of monozygous twins show indistinguishable banding patterns and the bands are inherited according to the Mendelian laws. Thus this procedure reveals informative fingerprints that can be used for individual identification, e. g. in paternity testing and in forensic applications. In most of these experiments 32P‐labelled probes were employed, yet the biotinylated oligonucleotide (GACA)4 produced results which were equivalent to those obtained by hybridization with the 32P‐labelled probe (GACA)4.
Subtyping of orosomucoid 1 (ORM1) by isoelectric focusing in agarose and polyacrylamide gels
Authors: Thymann Mariann , Weidinger Sebastian 
Published: May 2005

AbstractGenetic polymorphism of orosomucoid (ORM) has been demonstrated in several populations and comprises two structural gene loci, ORM 1 and ORM2. In Caucasians three common ORM 1 alleles have been shown, while the ORM 2 locus is almost monomorphic. ORM 1 phenotyping by isoelectric focusing in agarose or polyacrylamide gel combined with either print immunofixation or enzyme‐linked immunoblotting is described, and population and family data from Denmark and Southern Germany are given. It is proposed to use a different alpha‐numerical nomenclature for the phenotypes of the ORM 1 and ORM 2 systems.