bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of nonfunctional overexpression gene products using two‐dimensional gel electrophoresis with a narrowed pH range
Authors: Wöhl Thorsten , Klier Hannelore , Lottspeich Friedrich 
Published: May 2005

AbstractA commonly used technique in the analysis of yeast protein function is the overexpression of cloned genes. In the case that overexpression does not lead to an altered phenotype a stable synthesis of the protein has to be demonstrated. Here an example is shown where overexpression of the yeast HYP2 genes, coding for a hypusine‐containing protein, was alternatively analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) alone or by two‐dimensional (2‐D) gel electrophoresis, using the narrowed pH range of 4.5–5.4. The results of the SDS‐PAGE suggested a stable overproduction of HYP gene product, whereas 2‐D analysis revealed an accumulation of nonfunctional protein isoforms.
Tris‐dependent oxidative DNA strand scission during electrophoresis
Authors: Ray Trevor , Mills Andrew , Dyson Paul 
Published: May 2005

AbstractThe DNA of two Streptomyces species contains site‐specific labile modifications. During gel electrophoresis the DNA can undergo Tris‐dependent strand scission at the positions of these modifications. Our investigations into the nucleolytic activity which reacts with the modifications implicate a peracid derivative of Tris formed at the anode; the kinetics of production and decay of this activity were followed using both a DNA cleavage assay and a reduced methyl viologen assay to measure oxidant. Anode activation could be chemically mimicked by addition of peracetic acid to Tris buffers. We tested the DNA cleavage activity of several other compounds after anode or chemical activation; we used an analogue of Tris lacking a primary amine group and also several reagents known to promote DNA strand cleavage by amine‐catalysis at abasic sites. Anode generation of oxidant could be detected for compounds containing either hydroxyl or carboxyl groups. However, DNA cleavage activity correlated with oxidant formation only for those compounds also containing primary amine groups. These results support a mechanism of DNA strand scission at modification sites via concerted peracid‐mediated oxidative and amine‐catalysed reactions. The novel finding of Tris‐dependent formation of a long‐lived reactive oxidant at the anode suggests that this compound is unsuited as an electrophoresis buffer for certain biological macromolecules.
A preparative method for sequencing proteins and peptides: In situ gel staining with subsequent passive elution onto polyvinylidine difluoride membranes
Authors: Warlow Robert S. , Gooley Andrew , Rajasekariah Poornima , Oszarac Nesrin , Walls Ronald S. 
Published: May 2005

AbstractA preparative method for obtaining both N‐terminal and internal peptide amino acid sequences from purified proteins is reported. The methodology reliably yields high fidelity signal from between 14 to 30 residues per purified protein or peptide, with low backgrounds on amino acid analysis. The procedure relies on the use of in situ staining of proteins during preparative sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and the utilisation of microconcentrators to repeatedly concentrate small amounts of proteins onto a small polyvinylidene difluoride (PVDF) disc until sufficient amounts have been adsorbed so as to give a strong sequencing signal. The protein elution and subsequent adsorption can be monitored visually with a dye and the final product, a PVDF disc with the adsorbed protein or peptide, can be directly inserted into the automated amino acid sequencer.
The influence of proteins on silver staining of nucleic acids following polyacrylamide gel electrophoresis
Authors: Hilbert Friederike , Mayr Burkhard , Lackner Fritz , Bauer Friedrich 
Published: May 2005

AbstractThe influence, on silver staining, of proteins and restriction enzymes in polymerase chain reaction (PCR) products was studied in 12–20% polyacrylamide gels. For small DNA fragments (74, 41 and 33 bp) best results were achieved using 20% polyacrylamide gels. In 12% gels, restriction enzymes and also proteins used for PCR (bovine serum albumin, Taq‐DNA polymerase) interfere with silver staining of nucleic acids.
The Dukhin‐Deryaguin equation for the electrophoretic mobility in monovalent electrolytes with arbitrary ion mobilities
Authors: Egorova Elena M. 
Published: May 2005

AbstractThe Dukhin‐Deryaguin equation, used in the modern theory of electrophoresis for the calculation of electrophoretic mobility (EPM) in the region of double layer polarization, is known from literature [4–6] in its form (DD1) valid for the equal mobilities of the cation and anion in solutions of symmetrical electrolytes. Here we describe the other version of this equation (DD2), for arbitrary ion mobilities in 1:1 electrolytes. The EPMs calculated from this latter version are in good agreement with an exact computer solution [12]. The use of DD2 is illustrated in a series of EMP vs. lgC curves, calculated for selected examples of negatively charged lipid membranes. In addition, we describe two simplified versions of DD2, which are valid, respectively, for the high zeta potentials and when the electroosmotic component of ions' fluxes at the charged surface is neglected. Comparing DD2 with DD1 shows that the latter equation results in an error which may exceed the experimental dispersion of EPM values in the absence of specific ion binding. This error is reduced if the counter‐ion binding is not small; hence, DD1 may also be used in some cases for solutions with arbitrary ion mobilities.
Single‐step electrotransfer of reverse‐stained proteins from sodium dodecyl sulfate‐polyacrylamide gel onto reversed‐phase minicartridge and subsequent desalting and elution with a conventional high‐performance liquid chromatography gradient system for analysis
Authors: Fernandez‐Patron Carlos , Madrazo Joel , Hardy Eugenio , Mendez Enrique , Frank Rainer , Castellanos‐Serra Lila 
Published: May 2005

AbstractIsolation of proteins from polyacrylamide electrophoresis gels by a novel combination of techniques is described. A given protein band from a reverse stained (imidazol‐sodium dodecyl sulfate – zinc salts) gel can be directly electrotransferred onto a reversed‐phase chromatographic support, packed in a self‐made minicartridge (2 mm in thickness, 8 mm in internal diameter, made of inert polymeric materials). The minicartridge is then connected to a high‐performance liquid chromatography system and the electrotransferred protein eluted by applying an acetonitrile gradient. Proteins elute in a small volume (> 700 μL) of high‐purity volatile solvents (water, trifluoroacetic acid, acetonitrile) and are free of contaminants (gel contaminants, salts, etc). Electrotransferred proteins were efficiently retained, e.g., up to 90% for radioiodinated α‐lactalbumin, by the octadecyl matrix, and their recovery on elution from the minicartridge was in the range typical for this type of chromatographic support, e.g., 73% for α‐lactalbumin. The technique was successfully applied to a variety of proteins in the molecular mass range 6–68 kDa, and with amounts between 50 and 2000 pmol. The good mechanical and chemical stability of the developed minicartridges, during electrotransfer and chromatography, allowed their repeated use. This new technique permitted a single‐step separation of two proteins unresolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis due to their different elution from the reversed‐phase support. The isolated proteins were amenable to analysis by N‐terminal sequencing, enzymic digestion and mass spectrometry of their proteolytic fragments. Chromatographic elution of proteins from the reversed‐phase mini‐cartridge was apparently independent of the specific loading mode employed, i.e., loading by conventional loop injection or by electrotransfer.
Sodium chloride in separation medium enhances cell compatibility of free flow electrophoresis
Authors: Bondy Brigitta , Bauer Johann , Seuffert Ingrid , Weber Gerhard 
Published: May 2005

AbstractFree flow electrophoresis of cell suspensions in buffers containing sodium chloride was investigated using a modified procedure and the new apparatus Octopus PZE. The major methodical innovations are upward fluid flow margin buffers flowing through the electrophoresis chamber at both sides of a central cell suspension buffer, adjacent to the electrode membranes, and a sample injection device which focuses the cells hydrodynamically to the middle of the chamber thickness. Mononuclear leukocytes, suspended in a buffer containing 35 mM NaCl, could be fractionated with the same accuracy as by conventional free flow electrophoresis, operated with a single NaCl‐free chamber buffer. However, testing the vitality of separated cells with the help of the calcium indicator FURA2‐AM clearly demonstrated the biological importance of the presence of a minimum amount of sodium chloride during cell electrophoresis. Only if at least 35 mM NaCl were present could an undisturbed cytosolic Ca2+ metabolism be maintained for the time of a free flow electrophoresis cell separation experiment.
Horizontal gel electrophoresis with sample volumes up to 1.5 mL, using a discontinuous buffer system and automated apparatus
Authors: Chang Huan‐Tsung , Chrambach Andreas 
Published: May 2005

AbstractHorizontal gel electrophoresis has previously suffered from lack of an instrumental design that would allow for application of large sample volumes. A recently introduced commercial apparatus (HPGE‐1000, LabIntelligence) remedies that problem in application to gel electrophoresis with intermittent scanning of fluorescence by using the concentrator module of that apparatus as a stacking gel reservoir. By using this technique and a 3 mL stacking gel, protein samples of up to 1.5 mL yield bands in the horizontal resolving gel that are independent of sample volume in their width, area and migration rate. A remaining procedural problem relates to the apparent nonsimultaneity of dye and protein entrance into the resolving gel, which necessitates peak characterization by absolute rather than relative mobilities.
Direct detection of β‐1,3‐glucanase in plant extracts by polyacrylamide gel electrophoresis
Authors: Kalix Siegfried , Buchenauer Heinrich 
Published: May 2005

AbstractBy using carboxymethyl (CM)‐curdlan, a polysaccharide linked with the dye Remazol Brilliant Blue (RBB) as a substrate in polyacrylamide gels, the β‐1,3‐glucanase in plant extracts can be detected directly by native polyacrylamide gel electrophoresis. In contrast to the usually used procedures for the detection of glucanases, e.g., colorimetric assay, overlay technique, enzyme activity staining using laminarin as a substrate, this method is rapid and allows both the determination of the activity and the location of the relative position of the multiple forms of β‐1,3‐glucanases.
Amino acid compositions of proteins and their identities
Authors: Galat Andrzej , Bouet Françoise , Rivière Sylvie 
Published: May 2005

AbstractA critical overview is given on the application of amino acid composition data for the establishment of the protein's identity (amino acids composition vs. protein identity, the AAC‐PI method). Several criteria are used to measure the differences between the amino acid compositions of various proteins. The AAC‐PI method unambigiously identifies proteins which belong to the families with a high phylogenetic conservancy of their sequences. The identification of pure proteins can be accomplished with a relatively high level of confidence. The AAC‐PI method, however, sometimes needs the support of N‐terminal or internal sequencing of proteins since, alone, it cannot distinguish whether the lack of finding a candidate protein in protein data bases is because the investigated amino acid composition corresponds to an unknown protein or its processed form or because it is a sum of at least two protein components, or whether it is due to other experimental errors. The identification of a few new proteins such as “arginine‐rich protein”, macrophage migration inhibitory factor (MIF) and the preformed neurotrophic factor present in the calf brain cytosol is also reported.
Human blood platelet protein map established by two‐dimensional polyacrylamide gel electrophoresis
Authors: Gravel Patricia , Sanchez Jean‐Charles , Walzer Claude , Golaz Olivier , Hochstrasser Denis F. , Balant Luc P. , Hughes Graham J. , Garcia‐Sevilla Jesus , Guimon José 
Published: May 2005

AbstractTwo‐dimensional (2‐D) maps of cytosol and enriched‐membrane platelet proteins has allowed the identification of more than 25 spots by three different methods: matching of the platelet gels with other 2‐D reference maps, immunoblotting with chemiluminescence detection, and N‐terminal sequencing. Different G protein (guanosine triphosphate‐binding protein) subunits, cytoskeletal proteins, and proteins common to the human liver, red blood cells and plasma were identified. The two platelet protein maps presented here contribute to the project of identification of human cell and body fluid proteins. They may serve as working tools since platelets are popular models for the study of central nervous system neurotransmitter systems and stimulus‐response coupling mechanisms.
Two‐dimensional electrophoresis of human serum proteins modified by ampicillin during therapeutic treatment
Authors: Magi Barbara , Marzocchi Barbara , Bini Luca , Cellesi Carla , Rossolini Aldo , Pallini Vitaliano 
Published: May 2005

AbstractTwo‐dimensional electrophoretograms of serum proteins from ampicillintreated patients were analyzed by immunoblotting with an antiserum specific for penicilloyl groups. As expected, human serum albumin (HSA) was the main ampicilloylated serum component. Transferrin main form II was found to be the second most important component as regards immunoblotting intensity. Immunoreactive spots were present on the acidic side of the transferrin isoelectric series, suggesting a modification mechanism similar to that observed in HSA, i.e., acylation of basic amino acid residues. Several additional ampicilloylated spots were detected but could not be assigned. Their electrophoretic parameters were determined using internal standards. This is the first description of serum proteins other than HSA being modified by ampicillin in the course of routine therapeutic treatment.
Separation of the two most closely related isoenzymes of alkaline phosphatase by two‐dimensional electrophoresis
Authors: Otto Viviane I. , Fried Roman , Wiederkehr Felix , Hänseler Edgar 
Published: May 2005

AbstractSeminoma is the most frequent testicular germ cell tumor. While effective curative treatment of the disease is available today, there is to date no tumor marker suited for the diagnosis and follow‐up. Several authors have suggested that the germ‐cell‐specific isoenzyme of alkaline phosphatase (GCAP) might be valuable for this purpose. GCAP shows 98% sequence homology with the placental isoenzyme of alkaline phosphatase (PLAP). Both display a high degree of phenotypic heterogeneity. Until now all attempts to raise an antibody reacting specifically with GCAP have failed. Consequently there is no immunological assay that allows the measurement of GCAP in the presence of PLAP. Two‐dimensional electrophoresis with a sigmoid immobilized pH‐gradient of 3–10 for the first dimension makes it possible to differentiate clearly between these two closely realted isoenzymes. Additionally, it resolves their many phenotypic variants. This is of special interest, since malignant transformation affects the glycosylation patterns of many glycoproteins. For the detection of GCAP and PLAP in two‐dimensional electrophoresis it is essential to purify the raw tissue extracts thoroughly. A chromatographic method suited for this purpose is presented.
Electrophoretic and chromatographic differentiation of two forms of albumin in equilibrium at neutral pH: New screening techniques for determination of ligand binding to albumin
Authors: Bjerrum Ole J. , Bjerrum Morten J. , Heegaard Niels H. H. 
Published: May 2005

AbstractAnalysis of normal human serum by crossed hydrophobic interaction immunoelectrophoresis with Phenyl‐Sepharose revealed a biphasic appearance of the albumin peak. The molecular mechanism behind this apparent albumin heterogeneity was investigated. Analysis of defatted purified albumin showed that a major fraction bound to the Phenyl‐Sepharose and that addition of ligands (e.g. long‐chain fatty acids, bilirubin, sulfonamides and warfarin) before electrophoresis blocked this binding to different degrees. A quantitative relation between ligand binding and the amount of nonbinding albumin was found. Thus the technique might be suitable for screening of ligand binding to albumin. Analysis of serum samples from newborns with hyperbilirubinemia revealed a positive correlation between the fraction of the nonretarded albumin and the bilirubin concentration. By chromatography on Phenyl‐Sepharose, defatted albumin was separated into a binding and a nonbinding from and this technique was subsequently used to determine the kinetics of the intramolecular conversion. After rechromatography, each of the fractions could again be separated into two fractions, indicating the presence of an eqilibrium. By varying the passage time for albumin on the column or varying the period between the first and the second separation it was possible to calculate the conversion rates. The half‐life for the conversion was found to be as long as 1 ¼ h. It is the first time that a conformational change for albumin with such a long conversion time has been described experimentally.
Micropreparative one‐ and two‐dimensional electrophoresis: Improvement with new photopolymerization systems
Authors: Rabilloud Thierry , Vincon Mathilde , Garin Jerome 
Published: May 2005

AbstractTo improve the efficiency of one‐ and two‐dimensional electrophoresis for micropreparative purposes, the use of gels polymerized with other initiators than the standard N,N,N′,N′‐tetramethylethylenediamine (TEMED)/persulfate systems for sodium dodecyl sulfate electrophoresis has been investigated. We show here that the recently described photoinitiator system, composed of methylene blue, toluene sulfinate and diphenyliodonium chloride, leads to a decreased resolution. Resolution can be restored if methylene blue is replaced by riboflavin. Two‐dimensional electrophoresis with mg loadings of proteins has also been evaluated with these systems. Independently of the polymerization system, resolution for the first dimension is low with rod gels, increases with gel strips and is further improved when immobilized pH gradients are used. Here too, only the riboflavin/sulfinate/iodonium system results in a resolution that matches the one obtained with the standard TEMED/persulfate system. Gels polymerized with the riboflavin/sulfinate/iodonium system yield better results upon N‐terminal microsequencing after blotting than gels polymerized with the standard TEMED/persulfate system.
Forensic applications of a rapid, sensitive, and precise multiplex analysis of the four short tandem repeat loci HUMVWF31/A, HUMTH01, HUMF13A1, and HUMFES/FPS
Authors: Robertson James M. , Sgueglia Joanne B. , Badger Charles A. , Juston Ann C. , Ballantyne Jack 
Published: May 2005

AbstractA system of four short tandem repeat loci (HUMVWF31A, HUMTH01, HUMF13A1, and HUMFES/FPS) has been tested in co‐amplification with forensic (post‐mortem and post‐coital) DNA samples. Semiautomated DNA typing was employed to analyze polymerase chain reaction (PCR) products formed by extension of primers labeled with a fluorescent dye at the 5′‐terminus. Most DNA extracts could be typed, although a few required the addition of bovine serum albumin or a pretreatment by ultrafiltration in order to obtain sufficient signal for typing. Balanced signals for the alleles were obtained frequently across the loci, although preferential amplification of HUMTH01 was observed often with the forensic samples. Band splitting due to nontemplate nucleotide addition to the blunt ends of the amplimers was frequently detected for the DNA extracted from the forensic samples. A database was constructed for the African‐American population and compared with a Caucasian database. Few differences were observed across the two populations, except at the locus HUMTH01. The fluorescence‐based system facilitates large‐scale databasing, because the PCR products run off the gel, allowing more than one set of samples to be analyzed per run. Polyacrylamide gel reuse did not diminish genotyping accuracy.
Mass disasters: Rapid molecular screening of human remains by means of short tandem repeats typing
Authors: Corach Daniel , Sala Andrea , Penacino Gustavo , Sotelo Alicia 
Published: May 2005

AbstractHuman remains identification represents a challenging situation and constitutes a difficult task associated with mass disasters. The only highly efficient means for individual and family group reconstruction is that based on DNA typing. On July 18, 1994 an explosion destroyed the A.M.I.A. (Argentine Israeli Association.) Over 100 people died; however, the exact number of victims is still being investigated. Our Service received over 70 remains to be characterized by DNA typing in order to determine the number of victims and to try to reconstruct the family groups to which they belonged. DNA was extracted by a cetyltrimethylammonium bromide (CTAB) based protocol, a rapid molecular screening of all samples was carried out by multiplex STR amplifications including HUMTH01, HUMFABP, HUMHPRTB, HUMRENA4, HUMVWA. HUMFES/FPS and Y27H39LR. Samples with identical genotypes were HaeIII‐digested. Southern blotted and probed with YNH‐24 (D2S44). PH‐30 (D4S139). LH‐1 (D5S110) and MS‐1 (D1S7) for variable number of tandem repeats (VNTR) evaluation. The minisatellite variant repeat (MVR) approach was used in those cases in which band or profile shift were detected in Southern blot assays. Kinship between victims and putative relatives was initially evaluated by comparison of short tandem repeat (STR) profiles and then confirmed by VNTR with the above probes. The high identification efficiency attained in this case is, in part, supported by a previous experience, the DNA‐based molecular characterization of human remains caused by the explosion of the Israeli Embassy in Buenos Aires, March 1992.
DNA fingerprinting in silkworm Bombyx mori using banded krait minor satellite DNA‐derived probe
Authors: Nagaraju Javaregowda , Sharma Abhay , Sethuraman Balakathiresan N. , Rao G. V. , Singh Lalji 
Published: May 2005

AbstractThe genomic DNA from thirteen different ecotypes and inbred lines of silkworm, Bombyx mori, were analyzed by digesting with BstNI and HinfI restriction enzymes followed by hybridization with banded krait minor satellite DNA (Bkm)–2(8) minisatellite probe. The DNA fingerprinting revealed 9–31 discrete intense bands, some of which were ecotype/inbred line‐specific. Individual specific DNA fingerprints in two representative genotypes and their F1 hybrid offspring were also obtained. Individuals of a given parental line showed very similar profiles and the hybrid offspring showed the combined profile of both parents. The presence of bands specific to diapausing and non‐diapausing strains and to particular genotypes indicate their potential use for marker‐assisted breeding and varietal identification.