bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and identification of tyrosine‐phosphorylated proteins from B lymphocytes stimulated through the antigen receptor
Authors: Gold Michael R. , Yungwirth Tom , Sutherland Claire L. , Ingham Robert J. , Vianzon Daisy , Chiu Readman , Van Oostveen Inge , Morrison Hamish D. , Aebersold Ruedi 
Published: May 2005

AbstractThe activation of protein tyrosine kinases (PTKs) and subsequent tyrosine phosphorylation of cellular proteins is a critical initial signal in the response of eukaryotic cells to mitogens, differentiative signals, and other stimuli. A number of PTK substrates have been identified and many of these are components of signal transduction pathways that regulate cell function. However, the majority of proteins that are tyrosine‐phosphorylated in response to receptor signaling remain unidentified. As some of these unidentified PTK substrates may also be signal‐transducing proteins, their identification and functional characterization is an important objective towards understanding receptor signaling. We describe the development of a comprehensive and general process for the isolation and structural characterization of tyrosine‐phosphorylated proteins. The method involves enrichment by anti‐phosphotyrosine affinity chromatography, electrophoretic concentration and separation, and proteolytic fragmentation of individual purified phosphoproteins. Resulting peptide fragments are separated by microbore reverse‐phase high performance liquid chromatography (RP‐HPLC) and a portion of the eluted peptides are subjected to electrospray‐mass spectrometry (ES/MS) for accurate determination of peptide masses. Proteolytic fragmentation of a protein produces a characteristic set of peptide masses that can be used to rapidly identify the protein by searching databases containing the peptide mass “fingerprints” for all known proteins. The identity of the protein established by this method can be confirmed by sequence analysis of selected peptides. We have applied this procedure to the analysis of PTK substrates from B lymphocytes that have been stimulated through the B cell antigen receptor (BCR). Signaling by this receptor is involved in the generation of antibodies against foreign molecules (antigens). The BCR activates multiple PTKs which phosphorylate at least 30 different proteins. We have identified several of these tyrosine‐phosphorylated proteins, including Syk, a PTK that is known to be tyrosine‐phosphorylated in activated B cells. Thus, the procedure described here can be used to identify regulatory proteins of low abundance. The process consists of a logical succession of compatible steps that avoids pitfalls inherent to prior attempts to characterize low abundance phosphoproteins and should find wide use for the identification of tyrosine‐phosphorylated proteins in other cell types.
“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

AbstractWe 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

AbstractAn 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

AbstractThe 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

AbstractA 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

AbstractThe 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

AbstractA 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

AbstractElectrophoretic 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

AbstractHigh 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

AbstractA 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

AbstractThe 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

AbstractThe 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

AbstractHybridization 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

AbstractSomatic 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

AbstractUltrathin 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 gelsParts 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

AbstractWe 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

AbstractThe 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

AbstractA 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

AbstractElectrophoretic 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.