bluuebunny/crossref_metadata_2025 huggingface dataset:
“Spot transfer”, elution and comigration with known proteins allows accurate transferral of protein identifications between distinct two‐dimensional electrophoretic systems
Authors: Dean David P. , Cronan Melissa T. , Merenda Joseph M. , Gardner Joseph P. , Connelly Patricia A. , Celis Julio E.
Published: May 2005

We report a simple method, designated “spot transfer”, where known proteins are excised and eluted from a two‐dimensional (2‐D) gel run on one gel system to transfer identification of the proteins to a different 2‐D gel system by comigration with a comparable sample. In one experiment, 8 of 16 proteins eluted from an isoelectric focusing (IEF) 2‐D gel, of the format described for the Celis human keratinocyte database (Celis, J. E. et al., Electrophoresis 1993, 14, 1091–1198), were found to comigrate with proteins in a human T lymphoma (JURKAT) whole cell lysate run using the Millipore Investigator 2‐D system. The method should have general utility in allowing the exchange of protein identifications between investigators and could be used to standardize gel loci used in 2‐D gel protein databases.
Electrophoretic mobility profiles: New aspects of characterization and discrimination of biological surfaces
Authors: Budde Axel , Knippel Eberhard , Grümmer Gerald , Nitzsche Rolf
Published: May 2005

An advanced method of measuring and analyzing electrophoretic mobility profiles (electrophoretic fingerprints) was developed and for the first time applied to the cell surface. Dissociation constants calculated from a suitable mathematical model describe dissociation and, in part, adsorption processes on the surface and can thus be used for the characterization of complex processes on biological surfaces and the evaluation of small differences between cell surfaces.
On the use of dimensionless parameters in acid‐base theory: VI. The buffer capacities of equimolar binary mixtures of monovalent weak protolytes as compared to that of bivalent protolytes
Authors: Rilbe Harry
Published: May 2005

The general equation for the relative molar buffer capacity, earlier shown to be valid for bivalent acids, bases, and ampholytes, is shown to hold also for equi‐molar, binary mixtures of monovalent protolytes if only the parameter s = \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm}\sqrt {K_{\rm 1} ^\prime {\rm /4}K_{\rm 2} ^\prime} $\end{document} is exchanged for t = s + 1/4s. The same applies to the equations for the mean valence of the two classes of protolytes. As a consequence thereof, the titration and buffer capacity curves of a bivalent protolyte are identical with those of a monovalent protolyte with a pK′ value equal to \documentclass{article}\pagestyle{empty}\begin{document}${\rm}\sqrt {K_{\rm 1} ^\prime {\rm}K_{\rm 2} ^\prime} $\end{document} of the bivalent one (the isoprotic point of an ampholyte). For a hypothetical bivalent acid, base, or ampholyte with s = 1, ΔpK′ = log 4, this implies that the intrinsic rather than the hybrid dissociation constants are responsible for the titration and buffer capacity curves.
Molecular size/shape effects in the separation of the monosubstituted alkyl pyridines using capillary electrophoresis
Authors: Rowe Raymond C. , Wren Stephen A. C. , McKillop Andrew G.
Published: May 2005

A series of monosubstituted alkyl pyridines have been used to investigate the presence of molecular size/shape effects in capillary electrophoresis and the applicability of various descriptors in discriminating not only between derivatives of differing chain length but also between positional isomers. Although van de Waals' radii and Offord's parameters are extremely good descriptors in predicting the relative mobilities of the derivatives, a further descriptor involving length and/or diameter of the molecule is necessary for discrimination between the positional isomers.
Ogston gel electrophoretic sieving: How is the fractional volume available to a particle related to its mobility and diffusion coefficient(s)?
Authors: Slater Gary W. , Guo Hong L.
Published: May 2005

The Ogston‐Morris‐Rodbard‐Chrambach model (OMRCM) of gel electrophoresis assumes that the mobility μ of charged particles is proportional to the fractional volume (f) of the gel that is available to them. If the gel is random, as described by Ogston, the (semi‐log) Ferguson plot is the method of choice for analyzing experimental data since it permits an estimate of the gel's mean pore size to be made. However, the Ferguson plot is rarely linear; this is usually “explained” by the deformation of the anisotropy of the particle, the nonrandom or variable architecture or the gel, or the onset of some other migration mechanism. Many authors have refined this model, but the original assumption that μ ∝ f has not been seriously examined. Also, the model says nothing of the effect of the field intensity, the connectivity of the gel pores, nor anything about the diffusion coefficient. We have developed a Monte‐Carlo computer simulation algorithm to study the electrophoretic sieving of simple particles in gels. In this brief communication, we report important preliminary results which indicate that the basic assumptions of the OMRCM are wrong. We use a two‐dimensional periodic gel since the OMRCM becomes trivial in this case. Our results show that the relationship between f and μ is not the one assumed by the OMRCM. Moreover, we find that the Einstein relation between the diffusion coefficient and the mobility is not valid. This is due to the fact that the particles do not have a uniform probability of visiting the various sites that are available to them. We thus conclude that the Ferguson plot is intrinsically nonlinear; the curvature of the plot is, in fact, related to the intensity of the electric field as well as to the degree of randomness of the gel fibers.
Peptides as standards for denaturing isoelectric focusing
Authors: Watts Norman R. M. , Singh Rudra P.
Published: May 2005

A set of commercially available peptides suitable for use as standards in denaturing isoelectric focusing (IEF) is described. The peptides N‐procalcitonin fragment 1–57 (pI 3.98), Gln11‐amyloid β‐protein fragment 1–28 (pI 5.76), gastric inhibitory polypeptide (pI 7.14), parathyroid hormone fragment 1–34 (pI 8.64) and human β‐endorphin (pI 9.49) can be focused to their isoelectric point in the presence of 8 M urea and 2% Nonidet P–40, and subsequently fixed and stained in polyacrylamide gels. The peptides give a linear standard curve in close agreement with a slope determined with a surface pH electrode. Under the same conditions some proteins focus to positions significantly at odds with their theoretical isoelectric point. The origins of these discrepancies and the implications for the determination of isoelectric points of unknown proteins by denaturing IEF are discussed.
Electrophoretic conditions for high resolution citrus isozymes in polyacrylamide gel electrophoresis
Authors: King Brendon J. , Lee L. Slade , Rackemann R. Geoffrey , Scott Paul T.
Published: May 2005

Electrophoretic conditions including electrode and gel buffers, acrylamide concentration, use of stacking gels, voltage, current, and run time were investigated in order to produce isozyme bands of high resolution which would facilitate densitometric quantification of enzyme activity following polyacrylamide gel electrophoresis (PAGE). Electrode buffers which provided optimal conditions for gels stained for the isozymes of malate dehydrogenase (MDH), 6‐phosphogluconate dehydrogenase (6‐PGD), phosphoglucose isomerase (PGI), and shikimate dehydrogenase (SkDH) were 0.02 M Tris‐glycine, pH 8.5, 0.1 M sodium borate, pH 6.0, 0.1 M sodium borate, pH 8.7, and 0.07 M sodium borate, pH 7.0, respectively. A 0.5 M Tris‐HCl, pH 7.5, gel buffer was optimal for gels stained for the isozymes of 6‐PGD, PGI and SkDH. A 0.5 M Tris‐HCl, pH 8.5, gel buffer was best for gels stained for MDH. Stacking gels were found to be detrimental to enzyme activity and showed no improvement in resolution for any of the enzymes. Acrylamide concentration for gels stained for MDH were 8.7%, gels stained for 6‐PGD and PGI were 7.5%, while gels stained for SkDH had an acrylamide concentration of 5.0%. Higher concentrations above these levels caused a reduction and in some cases loss of band activity, while below this concentration there was a decrease in band resolution. Gels stained for MDH yielded best results when run for 6.5 h at a constant current of 5 mA/gel and an initial voltage of 40 V, gels stained for 6‐PGD were best after 10 h at an initial current of 8 mA/gel and a constant voltage of 140 V, gels stained for PGI were run for 22 h at an initial current of 9 mA/gel and a constant voltage of 34 V and gels stained for SkDH were run for 10 h at an initial current of 5 mA/gel and a constant voltage of 60 V. Triscitrate buffers used widely in Citrus and other taxons on both starch and polyacrylamide gels were found to be unsatisfactory. Higher molarity buffers with lower current and longer run times were found to provide superior resolution and band separation in comparison to lower molarity buffers with higher current and shorter run times. Zones of activity previously reported in Citrus but not in mandarin cultivars were revealed for both MDH and PGI. Our interpretation of the alleles for SkDH and 6‐PGD were not in agreement with those previously reported for the cultivars studied. These electrophoretic conditions provide isozyme bands of high resolution on PAGE, which will be suitable for densitometric analysis.
High‐performance capillary electrophoretic separation of proteins and peptides using a bonded hydrophilic phase capillary
Authors: Piccoli Giovanni , Fiorani Mara , Biagiarelli Beatrice , Palma Francesco , Vallorani Luciana , De Bellis Roberta , Stocchi Vilberto
Published: May 2005

High performance capillary electrophoresis (HPCE) was applied to the separation of protein and peptide mixtures with molecular masses ranging from 1300 to 96000 Da using a new bonded hydrophilic phase capillary, CElect‐P150. This coated capillary reduces the interaction between proteins and silanol groups in capillary walls, allowing a complete recovery of the proteins and peptides of interest. HPCE was also used for the analysis of a complex mixture of tryptic fragments and to monitor the process of enzymatic digestion. Moreover, using a CElect‐P150 capillary, highly reproducible analysis was possible without preconditioning the capillary with acid or basic solutions before each new analysis.
Analysis of immunoglobulin G using a capillary electrophoretic affinity assay with protein A and laser‐induced fluorescence detection
Authors: Lausch Ralf , Reif Oscar‐Werner , Riechel Peter , Scheper Thomas
Published: May 2005

A method for the rapid and sensitive determination of immunoglobulin G (IgG) in cultivation media by an affinity assay using capillary electrophoresis is presented. For that purpose we evaluated protein A conjugated with a fluorescent dye such as fluorescein diisocyanate or dichlorotriazinyl‐aminofluorescein as an affinity ligand. The ligand formed a fluorescing complex with immunoglobulin G in the sample and rapid separation from excess protein A was performed by capillary zone electrophoresis. However, only partial resolution of the zones was achieved when protein A as a whole molecule was utilized. In contrast, baseline resolution of the zones was obtained when recombinant fragments of protein A were used as affinity ligands. Immunoglobulin concentrations in the range of two orders of magnitude were determined. Due to the specifity of protein A for immunoglobulin G, analysis can be carried out even in the presence of high concentrations of other components and in cultivation media. Thus, the capillary electrophoretic affinity assay was successfully applied to monitor monoclonal antibodies in a cultivation process.
Application of capillary isoelectric focusing with absorption imaging detection to the quantitative determination of human hemoglobin variants
Authors: Wu Jiaqi , Pawliszyn Janusz
Published: May 2005

The capillary isoelectric focusing (CIEF)‐absorption imaging detector is a multicapillary separation instrument. Several protein samples can be separated and detected in a single run. The analysis time for one run is only 3 min. Because an imaging detector is used, the mobilization process of conventional CIEF is not necessary, eliminating such drawbacks as long analysis time and poor reproducibility in mobilization speed. Human hemoglobin variants were quantitatively determined using the instrument. The peak areas of the analytes were proportional to their concentrations in the concentration range of 0–200 μg/mL. Hemoglobin variant A2, which only comprises 2% of the whole hemoglobin, can be quantitatively determined with a standard deviation of less than 10%. For the high concentration variants, such as variant S, the deviation is less than 1%.
Effect of spike pulses on the orientation of the agarose gel matrix
Authors: Stellwagen John , Stellwagen Nancy C.
Published: May 2005

The orientation of the agarose gel matrix in two‐part, “stair‐step” electric fields has been studied by transient electric birefringence. Stair‐step electric fields are those in which a pulse of a given amplitude is immediately followed by a short, higher voltage “spike” pulse of the same polarity. A single stair‐step pulse orients the agarose gel matrix as though the two portions of the pulse were individually applied to the gel. However, a series of consecutive stair‐step pulses causes an anomalous increase in the amplitude of the birefringence, suggesting that increased numbers of agarose fiber bundles are orienting in the electric field. Spike pulses ≥ 10 V/cm appear to cause junction zone breakdown, freeing large numbers of agarose fiber bundles and microgel domains from the constraints of the gel matrix. The implications of these results for pulsed field gel electrophoresis are discussed.
The use of genomic DNA probes for in‐gel hybridization
Authors: Wöhl Thorsten , Brecht Michael , Lottspeich Friedrich , Ammer Hubert
Published: May 2005

Hybridization within agarose gels using oligonucleotide probes has been described in several publications; genomic DNA probes, however, have been used rarely and only with limited success. Here we present a simple and convenient procedure for in‐gel hybridization using radiolabeled genomic DNA fragments. The protocol was improved by the use of formamide in the hybridization as well as in the washing step. This method was compared with the conventional Southern blotting technique and was shown to produce good results in restriction pattern analysis, as well as in chromosomal localization with the help of pulsed field gel electrophoresis.
Analysis of thyroid stimulating hormone‐receptor mutations by temperature‐gradient gel electrophoresis
Authors: Koch Matthias , Wahl Richard , Seif Fritz J.
Published: May 2005

Somatic mutations in the genes for G‐protein‐coupled receptors which regulate intracellular levels of cyclic AMP have been found in several regions coding for the receptors of melanocyte‐stimulating hormone, adrenaline, luteinizing hormone, rhodopsin and thyrotropin. The mutations found in the thyroid‐stimulating hormone (TSH) receptor are restricted to cells of hyperfunctioning thyroid adenomas and other thyroid tumors. They are thought to lead to a constitutive activation of the receptor independent of TSH. We have developed a temperature‐gradient gel electrophoresis (TGGE) assay starting with the amplification of a part of exon 10 of the TSH‐receptor gene to screen tissue of thyroid tumours. TGGE allows the detection of point mutations even if there are only a few cells with somatic mutations in tissue sections.
Effective blotting of ultrathin polyacrylamide gels anchored to a solid matrix
Authors: Heukeshoven Jochen , Dernick Rudolf
Published: May 2005

Ultrathin polyacrylamide gels bound on glass plates or plastic sheets cannot be removed from their support without destruction. Therefore electrophoretic transfer methods are not applicable. We have developed a fast diffusion blotting procedure which is very simple and does not need any equipment like blotting chamber or power supply. Furthermore, no special buffer solutions are required. The method is universally applicable to ultrathin sodium dodecyl sulfate, native as well as isoelectric focusing polyacrylamide gels

Parts of this investigation were presented at ICES‐ELPHO 93, Sandefjord, Norway, June 2–4 1993

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Continuous focusing of biological particles by continuous immuno magnetic sorter: Technique and applications
Authors: Hartig Roland , Hausmann Michael , Cremer Christoph
Published: May 2005

We have developed a device for continuous deviation‐mode, open‐gradient fractionation of strongly magnetizable particles based on an electrophoresis counter‐flow chamber. A mixture of magnetically labeled and nonlabeled particles can be injected into a given continuously flowing chamber buffer. The particles pass the inhomogeneous magnetic field of the open‐gradient electromagnet in two narrow streams. According to the magnetic moments, induced by the magnetic field, magnetically labeled particles are deviated. The nonlabeled particles pass the magnetic field with negligible interaction. The deviated particles are focused into a stream that is completely separated from the streams of the nondeviated particles. The streams are fractionated by the counter‐flow technique and collected in different vials. A high sorting purity, depending only on the specificity of the antibodies or similar labeling techniques, and a high through‐put rate of up to 109 particles per hour was achieved. This was experimentally shown both by test particles and blood cells. The vital conditions of these blood cells were maintained by magnetic sorting.
The further construction of the two‐dimensional database of common human proteins
Authors: Burggraf Dorothe , Lottspeich Friedrich
Published: May 2005

The master two‐dimensional gel database (D. Burggraf et al., Electrophoresis 1992, 13, 729–732) [1] of common human proteins has been expanded to include detailed protein characteristics. Human cellular proteins from 5 cell lines and different cell organelles representing various tissues (muscle, nervous, connective, epithelial blood) and germ layers (ectoderm, endoderm and mesoderm), were separated by two‐dimensional gel electrophoresis (2‐DE). According to a recently developed algorithm, master gels of these different cells were established by computer‐aided image processing. An expanded map with protein‐chemical information of the polypeptides common to all human cells is shown. The synthetic, common human protein‐map represents 856 spots resolved with an accuracy of 4 cm/pI unit. The protein spots were characterized either by their isoelectric point, molecular mass, integrated intensity, background‐corrected optical density, spot area, or cellular distribution. About 80 proteins were further characterized and identified by protein name, amino acid composition analysis, N‐terminal sequencing, enzymatic digest and subsequent peptide sequencing. Additionally the proteins of the common human protein map were identified by Western blotting. Specific information regarding glycosylation and quantitation of expression levels after chemical, biological and mechanical stimulation is included in the database.
Two‐dimensional gel electrophoresis and immunoblotting of human serum albumin modified by reaction with penicillins
Authors: Marzocchi Barbara , Magi Barbara , Bini Luca , Cellesi Carla , Rossolini Aldo , Massidda Orietta , Pallini Vitaliano
Published: May 2005

A two‐dimensional gel electrophoresis and immunoblotting procedure has been developed to assess the level of modification by penicillins in human serum albumin. The procedure can be used in in vitro experiments and in clinical studies with sera from patients treated with penicillins.
Forensics on wild animals: Differentiation between otter and pheasant blood using electrophoretic methods
Authors: Miller Ingrid , Gutleb Arno C. , Kranz Andreas , Gemeiner Manfred
Published: May 2005

Electrophoretic techniques were used to prove illegal hunting of a protected species as small amounts of dried blood stains could be clearly identified as of otter origin. Species differentiation between the otter (Lutra lutra) and pheasant (Phasianus colchicus) was based mainly on small differences in electrophoretic mobilities of the respective albumins and hemoglobins in one‐dimensional separations. Additional differences in minor proteins of the two species were revealed by two‐dimensional electrophoresis. Electrophoresis thus also proved to be a valuable tool for forensic purposes.
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

A 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

The 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

A 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

The 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

The 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

Isolation 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

Free 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

Horizontal 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

By 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

A 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

Two‐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

Two‐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

Seminoma 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

Analysis 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

To 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

A 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

Human 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

The 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.
A region of Haemophilus influenzae Rd hybridizes with repetitive human DNA
Authors: Mody Rashna B. , Joshi Vasudha P.
Published: May 2005

A 17.7 kbp EcoR1 fragment of Haemophilus influenzae Rd (wild‐type strain) DNA containing repeats has been cloned together with the DNA repair gene uvr3. The region containing repetitive DNA was subcloned on a 13 kbp EcoR1 fragment (pHi13). The insert hybridizes with restriction enzyme digests of H. influenzae Rd and H. parainfluenzae and exhibits a repetitive pattern. Discrete bands superimposed upon a polydisperse background were observed when pHi13 hybridizes with restriction digests of human DNA. The hybridization patterns indicate that H. parainfluenzae and human DNA contain cross‐hybridizing sequences. The repeats of pHi13 are present on a 2.6 kbp PvuII fragment.
DNA fingerprinting for forensic identification: Some statistical issues
Authors: Majumder Partha P.
Published: May 2005

In DNA fingerprinting work, it is well known that, although fragment lengths of a sample are uncorrelated, measurement errors of fragment lengths are correlated. Unless the fragment length is very large, the measurement errors are directly proportional to fragment lengths. In the currently practiced matching procedures for forensic identification, these facts are ignored or inadequately used. Some of these issues are addressed in this study. Based on available empirical observations, an appropriate statistical model and techniques are proposed. These techniques lead to the definition and construction of elliptical match windows, instead of independent interval (linear) windows currently used. Simulation experiments indicate that the performance of the proposed procedure is superior to the current procedures. Further, the proposed procedure does not make use of some assumptions underlying the current procedures. Extensions of the procedure to utilize replicate measurements of crimescene and/or suspect's samples are indicated.
Application of DNA fingerprinting to the taxonomy of European blackberry species
Authors: Nybom Hilde , Kraft Thomas
Published: May 2005

Thousands of blackberry species (Rubus subgenus Rubus) have been described from Central and Northern Europe. These species are usually polyploid and pseudogamous. Since apomixis is only facultative, the species have retained the possibility to produce some offspring by sexual recombination. Hybridization of restriction‐enzyme‐digested DNA samples to the M13, (AC)/(TG) and 33.15 probes has yielded valuable information on the occurrence and distribution of genetic variation in Rubus. Intraspecific variation proved to be very restricted, with identical DNA fingerprints sometimes being exhibited by Swedish, Danish and German populations of the same species. By comparison, interspecific variation was usually pronounced, suggesting that most species have been derived through genetic recombination. Speciation through interspecific hybridization was demonstrated in one case. Several critical taxa were investigated; some of these proved to have unique DNA fingerprints, whereas others appear to be identical with previously described species. Evaluation of raspberry cultivars, obtained through several generations of vegetative propagation or with various in vitro techniques, failed to detect any DNA fingerprint variation, suggesting that the fingerprints are somatically stable. Blackberry seedlings, obtained by pollinating various species as well as interspecific hybrids with pollen from yet other species, were successfully evaluated with DNA fingerprinting to determine the relative proportions of the apomictic and sexual seed set.