bluuebunny/crossref_metadata_2025_split huggingface dataset:
Essential problems in quantification of proteins following colloidal staining with Coomassie Brilliant Blue dyes in polyacrylamide gels, and their solution
Authors: Neuhoff Volker , Stamm Reinhard , Pardowitz Iancu , Arold Norbert , Ehrhardt Wolfgang , Taube Dieter 
Published: May 2005

AbstractQuantitative determination of stained proteins following polyacrylamide gel electrophoresis (PAGE) is of increasing interest especially since computer‐aided densitometers have become available as well as recipes for sensitive and background‐free staining with Coomassie Brilliant Blue dyes. However, avoidance of separation artifacts is not the only essential prerequisite for quantitative evaluation. The local particle density of a protein in a given gel is of critical importance since it determines its stainability. Depending on local protein concentration, the dye binding to the same amount of a given protein differs considerably. Since the stainability of proteins using colloidal staining procedures, as with Coomassie Brilliant Blue dyes, is time‐dependent and, in addition, also dependent on the pore size of a given polyacrylamide gel used for PAGE, calibration curves for quantitative determinations have to be prepared in polyacrylamide gels of the same composition as used for PAGE. Staining conditions also have to be identical for calibration gels and gels under analysis. If, however, a set of calibration curves is prepared for different staining times, it is possible to calculate a generalized calibration curve, allowing for quantitative evaluation with flexible staining time. Furthermore, and in consequence of the implications due to particle density, quantitative determination via densitometry is only possible by determining the protein amount of each single measuring point (pixel) via its absorbance on the basis of a calibration curve. Since the particle density is inherent in a calibration curve, the final summation of the protein amount per pixel will give values close to reality.
Electrophoresis at elevated hydrostatic pressure of the multiheme hydroxylamine oxidoreductase
Authors: Masson Patrick , Arciero David M. , Hooper Alan B. , Balny Claude 
Published: May 2005

AbstractThe behavior of the multiheme protein hydroxylamine oxidoreductase (HAO) in polacrylamide gel electrophoresis was studied at hydrostatic pressures up to 3 kbar at 25°C. Due to the limited working volume of the high pressure vessel, the electrophoresis cells were miniaturized. A microcell which accommodates 6 capillary gel tubes is described. Between 1 bar and 1.5 kbar the enzyme did not undergo structural changes detectable in the gel system. At approximately 2 kbar the active form of the enzyme was partially dissociated. At higher pressures, the enzyme was converted to forms which were irreversibly inactive and had a higher apparent molecular mass, suggesting aggregation or denaturation.
Micro two‐dimensional gel electrophoresis of serum and testis esterases from different strains of mice
Authors: Abou‐Haīla Aīda , Fain‐Maurel Marcelle‐Anne 
Published: May 2005

AbstractTwo‐dimensional electrophoresis with time‐dependent polyacrylamide gradient gel electrophoresis (PAGGE) in the second dimension was applied to the separation of native molecular forms of esterases from serum and testis of four strains of mice (C57BL/6J, Swiss OF1, F1 hybrid derived from these two populations and Tfm). In Phast System, a modified pH 3–9 gradient, a linear 8–25 % gel gradient and a migration time corresponding to 300 Vh, were found to provide the best conditions for esterase analysis. About 70 esterase‐active fractions could be separated with good reproducibility. The variants were characterized by their pI (3.9–7.35), their relative mobility and the visual estimation of their susceptibility towards neuraminidase and different esterase inhibitors. In the two tissues, the distribution of the esterase variants corresponded to a 50–500 kDa molecular mass range of calibration proteins, but most of the serum and testis‐specific isoforms were confined to the 59–72 kDa range. All serum variants contained a terminal N‐acetylneuraminic acid residue, whereas only the testicular esterases in common with those in serum appeared sensitive to neuraminidase. Cholinesterases with a low relative mobility and carboxylesterases with a high relative mobility were detected in serum, while carboxylesterases accounted for the greatest part in the testis which also contained cholinesterases and acetylesterases. Minor interspecies differences were found between C57BL/6J and Swiss OF1 esterases. The expression of two variants which differed between these two species seemed intermediate for the hybrid originating form these two populations. Two new spots were detected in the two‐dimensional map of esterases from the strain bearing the Tfm mutation.
Synergistic induction of polypeptides by tumor necrosis factor and interferon‐gamma in cells sensitive or resistant to tumor necrosis factor: Assessment by computer based analysis of two‐dimensional gels using the PDQUEST system
Authors: Beresini Maureen H. , Sugarman Barry J. , Shepard H. Michael , Epstein Lois B. 
Published: May 2005

AbstractTumor necrosis factor (TNF) synergistically enhanced the antiproliferative activity of interferon‐gamma (IFN‐γ) in both TNF‐sensitive and TNF‐resistant variants of the cervical carcinoma line, ME‐180. TNF alone had no apparent effect on the levels of synthesis of individual proteins in either of these variant cell lines as assessed by computerized two‐dimensional gel analysis of cell lysates using the PDQUEST system. However, IFN‐γ enhanced the levels of 18 polypeptides and suppressed the levels of 10 polypeptides in both cell lines. When used in combination in both cell lines, TNF and IFN‐γ induced the synthesis of 10 polypeptides that were not induced by either agent alone. These synergistically induced polypeptides may be crucial to the mechanism of the synergistic antiproliferative action of TNF and IFN‐γ in ME‐180 cells.
Steady state electrolysis and isoelectric focusing
Authors: Hagedorn Rolf , Fuhr Günter 
Published: May 2005

AbstractThe properties of pH gradients formed by stationary electrolysis of weak mobile or fixed electrolytes are analyzed. The model uses the appropriate balance equations and those of chemical equilibria. It is shown how the equation of the current density has to be modified for considering that fraction of current that is associated with the diffusion of neutral buffer molecules within a pH gradient. Furthermore it is shown that the pH gradients themselves give rise to water production within the gradient and that essential properties of the steady state are related to chemical reactions between the electrolyte constituents. The differential equations describing the gradients of the concentration of a given component, the pH, conductivity and potential are explicitly formulated in relation to those reactions. The equations are solved numerically and the significance of the results for isoelectric focusing is discussed. The experimental conditions to reach shallow and smooth pH gradients exhibiting sufficient ionic strength are formulated.
The use of charge‐coupled devices in the quantitative evaluation of images, on photographic film or membranes, obtained following electrophoretic separation of DNA fragments
Authors: Boniszewski Zbigniew A. M. , Comley John S. , Hughes Barry , Read Christopher A. 
Published: May 2005

AbstractCharge‐coupled devices can provide an excellent means for the quantification of DNA band images on membranes and photographic film. However, proper consideration must be given to important parameters such as signal‐to‐noise ratio and resolution, especially in applications which demand the use of large format media. When employed in the direct imaging of chemiluminescent blots, the charge‐coupled device can provide equal or better sensitivity than that obtained by indirect methods using film, with the additional advantages of wide dynamic range and freedom from the vagaries of film processing.
A high resolution two‐dimensional gel electrophoresis and silver staining protocol demonstrated with nuclear matrix proteins
Authors: Cupo James F. , Lidgard Graham P. , Lichtman William F. 
Published: May 2005

AbstractAn improved two‐dimensional gel electrophoresis procedure has been developed utilizing isolated nuclear matrix proteins. The proteins of the cellular nuclear matrix are tissue specific. They are an example of a protein set whose two‐dimensional electrophoretic patterns afford much information of clinical significance. However, current two‐dimensional gel techniques were not completely satisfactory for the small amounts of protein present in tissue samples. There was a need for a two‐dimensional gel procedure which was capable of increased sensitivity and resolution and at the same time was reliable and reproducible. This has been accomplished by implementing several modifications to the current two‐dimensional gel procedures. In addition, changes were introduced in the silver staining process of the gels to increase the signal to background ratio. The overall procedure affects a dramatic increase in the resolution and clarity of the proteins visualized on two‐dimensional gels and is no more laborious than current techniques.
Combination of two‐dimensional gel electrophoresis with microsequencing and amino acid composition analysis: Improvement of speed and sensitivity in protein characterization
Authors: Eckerskorn Christoph , Lottspeich Friedrich 
Published: May 2005

AbstractHigh‐resolution two‐dimensional polyacrylamide electrophoresis (2‐DE) is commonly used as an analytical approach to resolve and detect most of the numerous protein species of an organism. However, the isolation of microgram amounts of protein in a 2‐DE spot in a form suitable for microsequence analysis and amino acid composition analysis is a key step in the chemical characterization of these proteins. With the development of chemically inert membranes it is now possible to retain proteins present in low quantities from the polyacrylamide matrix with high yields. The immobilized proteins are suitable for direct sequence analysis and amino acid composition analysis. The combination of protein chemical and electrophoretic techniques makes it possible to obtain chemical information from subpicomole quantities of protein, resulting in the availability of a new set of biologically important proteins for structural analysis. This paper summarizes the methods and strategies for the chemical protein analysis of 2‐DE spots in our laboratory.
Experimental and theoretical dynamics of isoelectric focusing: IV. Cathodic, anodic and symmetrical drifts of the pH gradient
Authors: Mosher Richard A. , Thormann Wolfgang 
Published: May 2005

AbstractThe production of anodic, cathodic and symmetrical drifts of a pH 3.5–10 gradient formed by isoelectric focusing in polyacrylamide gels is demonstrated experimentally by manipulation of the electrolyte concentrations. Experimental behavior is reproduced by computer simulation of a model mixture of 15 hypothetical carrier ampholytes whose pIs span the pH range 3 – 10. The mechanism which produces the drifts is elucidated and approaches to minimize such drifts are discussed. The data suggest why most experimentally observed drifts are cathodic.
Capillary zone electrophoretic separation of cyclodextrins with indirect UV photometric detection
Authors: Nardi Annalisa , Fanali Salvatore , Foret František 
Published: May 2005

AbstractA method for the separation of cyclodextrins by capillary zone electrophoresis with indirect detection is described. It is based on the formation of inclusion complexes with benzoate which simultaneously forms a UV absorbing constituent of the background electrolyte used for the separation.
Characterization of artifical liposomes with free‐flow electrophoresis
Authors: Kessler Reinhard , Manz Hans‐Jörg 
Published: May 2005

AbstractFree flow electrophoresis was used to examine the influence of active substances, lipid composition and preparation method on the surface charge of the liposomes. It is also possible to test the homogeneity of a liposome population with FFE.
Comprehensive two‐dimensional gel protein databases offer a global approach to the analysis of human cells: The transformed amnion cells (AMA) master database and its link to genome DNA sequence data
Authors: Celis Julio E. , Gesser Borbala , Rasmussen Hanne Holm , Madsen Peder , Leffers Henrik , Dejgaard Kurt , Honore Bent , Olsen Eydfinnur , Ratz Gitte , Lauridsen Jette B. , Basse Bodil , Mouritzen Solveig , Hellerup Marianne , Andersen Annette , Walbum Else , Celis Ariana , Bauw Guy , Puype Magda , Damme Jozef Van , Vandekerckhove Joël 
Published: May 2005

AbstractA total of 3430 polypeptides (2592 cellular; 838 secreted) from transformed human amnion cells (AMA) labeled with [35S]methionine were separated and recorded using computer‐aided two‐dimensional (2‐D) gel electrophoresis. A master 2‐D gel database of cellular protein information that includes both qualitative and quantitative annotations has been established. The protein numbers in this database differ from those reported in an earlier version (Celis et al. Leukemia 1988,2, 561–602) as a result of changes in the scanning hardware. The reported information includes: percentage of total radioactivity recovered from the gels (based on quantitations of polypeptides labeled with a mixture of 16 14C‐amino acids), protein name (including credit to investigators that aided identification), antibody against protein, cellular localization, (nuclear, 40S hnRNP, 20S snRNP U5, proteasomes, endoplasmic reticulum, mitochondria, Golgi, ribosomes, intermediate filaments, microfilaments and microtubules), levels in fetal human tissues, partial protein sequences (containing information on 48 human proteins microsequenced so far), cell cycle‐regulated proteins, proteins sensitive to interferons α, β, and γ, heat shock proteins, annexins and phosphorylated proteins. The results presented should be considered as the initial phase of a joint effort between our laboratories to undertake a general and systematic analysis of human proteins. Using this integrated approach it will be possible to identify phenotype‐specific proteins, to microsequence them and store the information in the database, to identify the corresponding genes, to search for homology with previously characterized proteins and to study the function of groups of proteins (pathways, organelles, etc.) that exhibit interesting regulatory properties. In particular, the 2‐D gel protein database may become increasingly important in view of the concerted effort to map and sequence the entire human genome.
The MRC‐5 human embryonal lung fibroblast two‐dimensional gel cellular protein database: Quantitative identification of polypeptides whose relative abundance differs between quiescent, proliferating and SV 40 transformed cells
Authors: Celis Julio E. , Dejgaard Kurt , Madsen Peder , Leffers Henrik , Gesser Borbala , Honore Bent , Rasmussen Hanne Holm , Olsen Eydfinnur , Lauridsen Jette B. , Ratz Gitte , Mouritzen Solveig , Basse Bodil , Hellerup Marianne , Celis Ariana , Puype Magda , Damme Josef Van , Vandekerckhove Joel 
Published: May 2005

AbstractA new version of the MRC‐5 two‐dimensional gel cellular protein database (Celis et al., Electrophoresis 1989, 10, 76–115) is presented. Gels were scanned with a Molecular Dynamics laser scanner and processed by the PDQUEST IITM software. A total of 1895 [35S]methionine‐labeled cellular polypeptides (1323 with isoelectric focusing and 572 with nonequilibrium pH gradient electrophoresis) are recorded in this database, containing quantitative and qualitative data on the relative abundance of cellular proteins synthesized by quiescent, proliferating and SV40 transformed MRC‐5 fibroblasts. Of the 592 proteins quantitated so far, the levels of 138 were up‐or down‐ regulated (51 and 87, respectively) by two times or more in the transformed cells as compared to their normal proliferating counterparts, while only 14 behaved similarly in quiescent cells. Seven MRC‐5 SV40 proteins, including plastin and two interferon‐induced proteins, were not detected in the master MRC‐5 images. The identity of 36 of the transformation‐sensitive proteins whose levels are up or down regulated by two times or more was determined and additional information can be transferred from the master transformed human epithelial amnion cells (AMA) database (Celis et al., Electrophoresis 1990, 11, 989–1071) for those polypeptides of known and unknown identity that have been matched to AMA polypeptides. As more information is gathered in this and other laboratories, including data on oncogene proteins and transcription factors, this comprehensive database will outline an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways and cytoskeletal systems that may be directly or indirectly involved in properties associated with the transformed state.
A convenient gel holder for preparative electrophoretic separation of aggregated bacterial proteins
Authors: Groschup Martin H. , Boschwitz Jeff , Timoney John F. 
Published: May 2005

AbstractA simple custom‐made gel holder for preparative SDS‐PAGE to separate aggregated bacterial antigens is described. The gel holder fits easily into commercially available gel electrophoresis apparatus and proteins or peptides are collected in a stream of distilled water through a channel in the gel. The performance of the device was illustrated by the successful separation and purification of fragments of aggregated Streptococcus equi M protein and of Erysipelothrix rhusiopathiae protective antigens. Yields of up to 1.2 mg per run were obtained. The purified proteins retained immunological reactivity and were of sufficient purity for amino acid compositional and sequence analysis.
Information on DNA conformation derived from the Ferguson plot of DNA fragments of up to 9 kb in size, using polyacrylamide gel electrophoresis in a discontinuous buffer system
Authors: Orbán László , Chrambach Andreas 
Published: May 2005

AbstractThe Ferguson plot in polyacrylamide gel electrophoresis (PAGE) (15 %CDATD, moving boundary electrophoresis buffer system operative at pH 8.9, 4°C, 8 mA/cm2 of gel) of DNA fragments up to 9.4 kb in size was found to exhibit a linear segment at polyacrylamide concentrations starting at 3 %T and undergoing a gradual transition into a concave segment at higher gel concentrations, confirming previous findings by Stellwagen [1]. The larger the DNA, and the higher the gel concentration, the less extended the linear and the more extended the concave segment of the plot. The lowest %T of the linear range for DNA in polyacrylamide remains unknown since mobilities at nongelling concentrations below 3%T have not as yet been measured. As previously suggested [1, 2], the transition from the linear to the concave segment corresponds to that from the randomly oriented DNA to the anisotropically stretched “reptating” DNA. For a DNA of 9.4 kb in size, the end of the linear range of the Ferguson plot can be extended from 3.5 to 5 %T when 15 % DATD rather than 2.5 % Bis is used to crosslink the polyacrylamide. Increasing the temperature of PAGE from 4°C to 25 and 50°C widens the linear segment progressively, indicating an increasingly random orientation with rising temperature. When current density is increased from 8 to 40 mA/cm2, the concave curvature of the Ferguson plot of DNA 1 to 9.4 kb in size decreases, suggesting a transition from a “reptating” to a randomly distributed molecule, due to increased Joule heat. Increasing the ionic strength from 0.01 to 0.04 M similarly appears to lead to a relative linearization of the Ferguson plot, suggesting a similar effect of Joule heat. Prestaining of DNA with ethidium bromide results in a decrease of both its size and its free mobility. This effect is exacerbated as the size of DNA increases from 0.1 to 9.4 kb.
Computer‐assisted statistical analyses of enzyme and ribosomal DNA electrophoretic polymorphism in Yersinia
Authors: Picard Bertrand , Denise Jean‐Baptiste , Decoux Guy , Goullet Philippe 
Published: May 2005

AbstractThe intra‐ and inter‐species differentiation of 90 strains of Yersinia belonging to six species were studied independently by computer‐assisted statistical analysis of data from enzyme electrophoretic polymorphism and ribosomal DNA (rDNA) restriction fragment length polymorphism. Two corresondence analyses (CA) demonstrated the concordance between the bacterial classification obtained from enzymatic and genomic data. This concordance was reinforced by an algorithm of the correspondence established between the two dendrograms drawn from previous computations. Comparison of CA with similarity analysis (also called “numerical taxonomy”) indicated that the intra‐ and inter‐species differentiation obtained by the two methods are similar. The advantage of CA is that it gives a synthetic geometrical representation of the results (factorial planes), displaying both the main features of the clusters of strains (location and dispersion) and their essential character (i.e.: enzyme electrophoretic variant, rDNA fragment size).
Application of silver staining to the rapid typing of the polymorphism of HLA‐DQ alleles, by enzymatic amplification and allele‐specific restriction fragment length polymorphism
Authors: Ju Li‐Ya , Gu Xue‐Fan , Larger Etienne , Krishnamoorthy Rajagopal , Charron Dominique 
Published: May 2005

AbstractA rapid and highly sensitive silver staining method, originally developed for the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA‐DQA1 and DQB1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their size separation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA‐DQ alleles of the genomic DNA. The in ensity of staining of digested PCR‐amplified DNA is linear from at least 8 to 18 ng for fragments of lengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele‐specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA‐DQ alleles at the nucleotide level in the clinical typing laboratory.
Serum amyloid A protein in very low density – and high density lipoproteins during the course of acute myocardial infarction
Authors: Feussner Giso , Schuster Markus , Ziegler Reinhard 
Published: May 2005

AbstractIn a preceding paper [1] we reported on the detection and characterization of human serum amyloid A protein (SAA) in very low density lipoproteins (VLDL) and high density lipoproteins (HDL) of patients after acute myocardial infarction. Here we describe the time course of the occurrence of SAA in VLDL and HDL in the postinfarction period. SAA reached its maximum in VLDL d HDL approximately 53 h after the acute event. At the peak of the acute‐phase response, SAA comprised as much as 38% of the total apoproteins of VLDL and HDL. SAA appeared at the same points in time and with nearly the same concentrations in VLDL and HDL. We conclude that SAA is not exchanged in plasma between lipoproteins of different densities and that this protein is secreted on its own by hepatocytes and not as a part of an already constituted lipoprotein particle.