bluuebunny/crossref_metadata_2025_split huggingface dataset:
Long‐term storage of free and polyacrylamide gel‐bound Immobiline chemicals
Authors: Pietta Piergiorgio , Pocaterra Enrica , Fiorino Antonio , Gianazza Elisabetta , Righetti Pier Giorgio 
Published: May 2005

AbstractThe stability with time of Immobiline solutions and of pre‐cast Immobiline gels has been investigated, for the former by high pressure liquid chromatography and for the latter by focusing a set of marker proteins. Temperature seems to affect these chemicals most: whatever degradation occurs at 20°C within a few weeks' time, is operative at 60°C within a few hours' time. These buffers are altered by pH in different ways: acidic Immobilines (pKs 3.6 and 4.6) are extensively degraded around neutrality (pH 6) while basic species (especially pK's 8.5 and 9.3) are massively destroyed in alkaline solutions (pH 9). Acidic pH values (pH 3–4) ensure maximum stability for all Immobiline chemicals. In terms of medium‐term storage of pre‐cast gels, wet matrices exhibit least degradation if titrated to pH 4. Formic acid is the best titrant since it can be efficiently removed during the focusing step with formation of the narrowest salt‐front at the anode. Dry matrices have much longer stability (>2 months) but should be stored with traces of humidity to prevent the cracking and peeling‐off from the plastic support which occurs at zero relative humidity.
Quantitative measurements of fluorescently‐stained DNA in gels from photographic images
Authors: Willis Charles E. , Holmquist Gerald P. 
Published: May 2005

AbstractPhotography under ultraviolet illumination is used routinely to record the results of gel electrophoresis of DNA when a fluorescent dye, which binds to DNA, is present in the gel. The distribution of DNA in the gel can be determined accurately from the photographic negative when the absorbance is interpreted according to the nonlinear film darking function, when the contrast of the film is calculated, and when the images are corrected for uneven ultraviolet transillumination. The contrast can be calculated from the images of a fluorescent plastic intensity standard. The local intensity of the unbound‐dye fluorescence can be used to correct images for uneven illumination. Using this approach, relative abundances of DNA fragments, compared within each electrophoretic track, were measured within an average error of 6.6% of the expected value with no evidence of systematic error. Quantitative comparisons were valid anywhere on the film for a 50:1 range of intensities, with errors distributed normally with a standard deviation of ± 10 % of the expected value.
A comparison of two‐dimensional gel electrophoresis methods for analysis of rat serum proteins following dimethylformamide exposure
Authors: Marshall Thomas , Williams Katherine M. , Vesterberg Olof 
Published: May 2005

AbstractFour two‐dimensional electrophoresis methods, each incorporating different protein denaturing/dissociating conditions, have been used to detect changes in rat serum proteins following exposure to dimethylformamide. The methods were evaluated on the basis of (i) their ability to detect changes, (ii) the resolution achieved and (iii) the response of resolution to the use of narrow Ampholine pH ranges in the first dimensional isoelectric focusing gels. All methods reveal minor but reproducible protein changes in response to dimethylformamide. However, high resolution two‐dimensional electrophoresis of the completely dissociated polypeptides reveals most changes and the resolution of this method is most improved by the use of Ampholines of narrow pH range. In general, the number of serum components detected and the resolution achieved is related to the severity of the protein denaturing/dissociating conditions employed. The significance of this observation in relation to others factors likely to influence the choice of two‐dimensional method is discussed.
Comparison of focusing in buffers and synthetic carrier ampholytes for use in the first dimension of two dimensional polyacrylamide gel electrophoresis
Authors: Burghes Arthur H. M. , Patel Ketan , Dunn Michael J. 
Published: May 2005

AbstractBuffer focusing using the mixture Polybuffer 47 (Polysciences) has been tested for the separation of proteins under denaturing conditions to assess its potential for use as the first dimension in two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). a relatively simple pattern, with the proteins confined to certain regions of the gel, was observed when [35S]methionine‐labelled fibroblasts were separated both by 2D‐PAGE and by one‐dimensional isoelectric focusing (IEF) using Polybuffer 47. When Polybuffer 47 and synthetic carrier ampholytes were mixed, the latter were also found to concentrate into regions corresponding to those occupied by the proteins. The addition of synthetic carrier ampholytes resulted in separation of proteins within these regions. Diagonal IEF gels of synthetic carrier ampholyte versus buffer focusing showed a “step” pattern, suggesting that buffer focusing resulted in concentration of proteins in particular regions. Diagonal IEF gels using synthetic carrier ampholytes in both dimensions verified that protein‐ampholyte interactions are not a major source of artefacts in such gels. When mixtures of Polybuffer 47 and synthetic carrier ampholytes were used, there was some extension of the pH gradient at both ends. We conclude that Polybuffer 47 alone is not suitable for use in the first dimension of high‐resolution 2D‐PAGE separations.
Identification map of human plasma proteins: Micro two‐dimensional electrophoresis followed by multiple immunoreplica technique
Authors: Manabe Takashi , Takahashi Yuko , Higuchi Noriko , Okuyama Tsuneo 
Published: May 2005

AbstractHuman plasma proteins were subjected to parallel micro two‐dimensional polyacrylamide gel electrophoresis in the absence of denaturing agents. Forty nitrocellulose blots were obtained from eight slab gels within one hour of electroblotting time, employing an apparatus designed to obtain multiple replicas from micro slab gels. The locations of 31 human plasma proteins were determined on these blots using specific antisera, and an “identification map” was prepared. Differences of protein location between plasma and serum were also demonstrated.
The engineering design of continuous electrophoresis
Authors: Andrews Graham F. , Fonta Jean‐Pierre 
Published: May 2005

AbstractIn the design of continuous electrophoresis devices, the main variables are the gap width, the flow rate and inlet temperature of the buffer, the electric field strength, the point of injection of the proteins, and the orientation of the flow to the gravitational field. Best values for many of these can be derived from first principles by requiring that the flow be stable, and that streak spreading be minimized. The result is a downflow device, with larger gap width than is usual, warm inlet buffer solution, and protein injection slightly downstream of the start of the electrodes. If cooling is improved by increasing the thermal conductivity of the cooling walls, the gap width must be reduced and the buffer flow increased. The performance possible with perfect cooling walls is similar to that obtained in space because, in both cases, the limiting factor is not thermal convection but the high temperature generated at the mid‐point between the cooling walls.
An isotachophoretic analysis of the interaction of bilirubin and biliverdin with bovine serum albumin
Authors: Oefner Peter , Csordas Adam , Bartsch Georg , Grunicke Hans 
Published: May 2005

AbstractThe binding sites of non‐defatted and fatty acid‐free bovine serum albumin for bilirubin and biliverdin were investigated by means of analytical capillary isotachophoresis. In addition, the effect of oleic acid on the binding of bilirubin was examined with the same analytical tool. Albumin‐bilirubin complexes with increasing molar ratios of bilirubin can be identified as a separately migrating zone adjacent to the zone of free bilirubin. At pH 9 there are 3–4 high‐affinity binding sites for bilirubin and, possibly, several weaker binding sites. Fatty acid‐free and non‐defatted albumin exhibit almost identical binding properties with regard to bilirubin. Under the same conditions non‐defatted albumin shows only one high‐affinity binding site for biliverdin. The analysis of fatty acid‐free albumin‐biliverdin complexes reveals additional weak binding sites, which indicates that the physiological loading of albumin with ligands interferes only with the low‐affinity binding of biliverdin. At sufficiently high concentrations, oleic acid even competes for the first high‐affinity binding site of bovine serum albumin for bilirubin. There is a linear relationship between the zone length and the molar concentrations of free ligands (bilirubin, biliverdin). The differential binding characteristics of bilirubin and biliverdin indicate that the reduction of biliverdin yields a molecule which has a higher affinity for albumin; this, however, results in an inferior water solubility at physiological pH values. Owing to high resolution and quantification, capillary isotachophoresis is a powerful tool for screening endogenous and exogenous compounds with regard to their effect on the bilirubin transport capacity of albumin.
Ultrathin‐layer isoelectric focusing of the photoaffinity labeled benzodiazepine receptor in calf brain: A new and simple method for the study of integral membrane proteins
Authors: Lentes Klaus Ulrich , Friedl Waltraut , Hebebrand Johannes , Propping Peter 
Published: May 2005

AbstractA new method for analytical isoelectric focusing of the benzodiazepine receptor in calf brain is described. Brain membranes are photoaffinity labeled with a specific radioligand, solubilized in 9 M urea and separated on 150 μm ultrathin polyacrylamide gels containing 8 M urea. The method combines high resolution with speed and versatility. It may be generally applicable for isoelectric focusing of integral membrane proteins in genetic studies.
Sensitive alkaline phosphatase linked secondary antibody system for detection of group specific component after isoelectric focusing on 250 μm thick reusable immobilized pH‐gradients
Authors: Pflug Werner 
Published: May 2005

AbstractA sensitive secondary antibody system was developed for detection of small amounts of group specific component (Gc). For the first antibody step after focusig, a comparison between immunofixation on cellulose acetate membranes and capillary blotting on nitrocellulose was made. The sensitivity of the first is much better than that of the latter. The method allowed the detection of about 150 picograms of Gc in 1:12 800 diluted serum samples. Even in six‐year‐old badly soluble bloodstains kept at room temperature, Gc could be phenotyped. The 250 μm thick immobilized pH‐gradient gels for focusing of Gc can be used repeatedly after washing and drying to the initial weight without destroying the pH gradient.
Urine analysis by two‐dimensional gel eletrophoresis with isoelectric focusing in immobilized pH gradients in the first dimension
Authors: Gianazza Elisabetta , Astrua‐Testori Silvia , Righetti Pier Giorgio , Bianchi‐Bosisio Adriana 
Published: May 2005

AbstractUrine analysis by two‐dimensional gel electrophoresis with isoelectric focusing in immobilized pH gradients in the first dimension allows the loading of high sample volumes and/or of large amounts of salts. When applied to urinary protein analysis, it makes it possible to apply up to 1 mL of dialyzed, unconcentrated sample or up to 350 μL of unprocessed urine.
HERMeS: A second generation approach to the automatic analysis of two‐dimensional electrophoresis gels. Part III: Spot list matching
Authors: Vincens Pierre , Tarroux Philippe 
Published: May 2005

AbstractOne of the most critical steps in two‐dimensional gel analysis is the comparison of spot lists. In this paper, we describe our approach to the matching problem. We propose a method based on syntactic pattern recognition techniques, which differs from previously used methods and offers a major improvement to cope with discontinuous geometric distortions. The method is based on non‐metric considerations and describes the pattern observed on a given gel in a non‐geometric way. This approach leads to the definition of finite automatons which can be used to automatically find the presence of a given pattern on a gel. The program uses a technique of clause generation related to artificial intelligence techniques. A set of rules is applied to the definition of homologousspots, and probability coefficients are computed to compare and to chose between different pairing hypotheses. Once matching has been achieved between a set of gels, the construction of a master gel which summarizes the information obtained is allowed. Two types of masters can be used. The first one summarizes the information between spots lists belonging to the same experiment and includes intensity considerations, while the second one summarizes the information resulting from different gels and does not include spot intensity and volume. Masters are written and known by the system as a spot list. This ability allows them to be handled just like spot lists and, using them, to perform all the actions available to the system. This approach is compared with a more classical one based on geometric corrections. The use of probability coefficients is also discussed and extended to their application to multiple matching and further utilization of masters.
Systematic analysis of serum lipoproteins and apolipoproteins by a combined technique of micro two‐dimensional electrophoresis
Authors: Manabe Takashi , Visvikis Sophia , Steinmetz Josiane , Galteau Marie M. , Okuyama Tsuneo , Siest Gérard 
Published: May 2005

AbstractLipoproteins in human serum and in lipoprotein fractions obtained by sequential ultracentrifugation of serum were analyzed by combining three types of micro two‐dimensional electrophoresis (2‐DE) technique to obtain systematic information on lipoproteins and apolipoproteins. The samples were first analyzed under non‐denaturing conditions of electrophoresis to characterize the physicochemical properties of low‐density lipoproteins (LDL) and high‐density lipoproteins (HDL) under physiological conditions. The samples were then analyzed, employing isoelectric focusing without denaturants in the first dimension and sodium dodecyl sulfate (SDS) electrophoresis in the second dimension, to study the process of dissociation of lipoproteins to apolipoproteins. The HDL's dissociated into their constituent apolipoproteins, showing that the charge heterogeneity of HDL is caused by the heterogeneous apolipoprotein content. LDL dissociated into apo B‐100 and minor components. In the third type of micro 2‐DE, employing urea and Nonidet P‐40 in the first dimension and SDS in the second dimension, the lipoproteins were dissociated into their apolipoproteins during the steps of sample treatment, to obtain the information on the contnt of apolipoprotein peptides and their genetic alleles. The combined micro 2‐DE technique will be useful for the systematic analysis of lipoproteins and apolipoproteins in hypo‐ and hyperlipoproteinemic patients.
Influence of flow and diffusion on protein separation in a continuous flow electrophoresis cell: Computation procedure
Authors: Biscans Béatrice , Alinat Philippe , Bertrand Joël , Sanchez Victor 
Published: May 2005

AbstractContinuous flow zone electrophoresis is an efficient method for the non‐destructive separation of biological materials in a flowing film of buffer solution in which the constituents of a sample are separated according to their electrophoretic mobilities, under the influence of an electric field. This paper deals with modeling of flow structure of the buffer solution taking into account the effect of electroosmosis, Joule heating and thermal free convection and the forced convection of axial flow. Modeling of diffusion is examined in a second part. The modeling equations and the corresponding boundary conditions are solved by finite difference methods. The various parameters affecting the quality of the fractionation are analyzed.
Purification and characterization of three forms of class III alcohol dehydrogenase
Authors: Valkonen Kaija H. , Goldman David 
Published: May 2005

AbstractWe have resolved and characterized three forms of human and rat hepatic class III alcohol dehydrogenase. Separations were carried out in narrow immobilized pH gradients. Both in humans and rats the three forms were visualized by enzyme staining with cinnamol, but not with ethanol. They were insensitive to the inhibitory effect of pyrazole. The isoelectric points were approximately from 6.3–6.4, from 5.9–6.0 and 5.6. Each electroeluted enzyme extract, purified further by analytical isoelectric focusing over the pH range from 5–6 or 6–7, revealed a single band by enzyme and silver staining and by Western blotting followed by avidin‐biotin staining. Polyacryl‐amide gel electrophoresis in the presence of sodium dodecyl sulfate of each extract revealed a single molecular mass species corresponding to class III alcohol dehydrogenase (ADH). All forms of class III alcohol dehydrogenase were recognized by antisera raised against total class III ADH.
Flattening and/or expanding of pH gradients in isoelectric focusing gels exemplified with PhastSystem
Authors: Hackler Rolf , Kleine Tilmann O. 
Published: May 2005

AbstractA simple method of flattening and/or expanding of pH gradients in isoelectric focusing is described for any pH interval desired: to modify pH gradients near one electrode a paper strip soaked with carrier ampholytes is applied onto the gel close to the opposite electrode. In order to flatten central parts of pH intervals paper strips are applied onto the gel at both electrodes. Conditions and criteria (e.g. amount and pH intervals of carrier ampholytes, width and localization of the paper strip, separation period) for optimization are presented with PhastSystem using ready‐made gels with three different pH intervals and pI marker proteins (Pharmacia). Examples utilizing erythrocyte lysates are presented.
Genetic polymorphism of human α2HS‐glycoprotein: Characterization and application to forensic hemogenetics
Authors: Yuasa Isao , Umetsu Kazuo 
Published: May 2005

AbstractHuman α2HS‐glycoprotein (AHSG) phenotypes were studied by isoelectric focusing of native and desialyzed samples followed by passive immunoblotting. At present a total of 15 different alleles could be distinguished. The usefulness of AHSG polymorphism in the field of forensic hemogenetics was demonstrated by markedly high variability at AHSG locus, reliability of phenotyping results and stability in blood stains.
Moving and stationary boundaries in immobilized pH gradients
Authors: Bjellqvist Bengt , Linderholm Marie , Östergren Karin , Strahler John 
Published: May 2005

AbstractThe relations describing the concentration changes at moving boundaries in a medium containing bound, buffering group are derived for a system which, except for hydrogen and hydroxyl ions, contains one anionic and one cationic mobile constituent. The relations found have been used to calculate concentrations and conductivities in zones developing in immobilized pH gradients. Assumptions used in the calculations as well as conductivity ratios between zones have been experimentally controlled and were found to reasonably agree with expectations. It is also shown how difference in transference numbers between sample droplet and gel will cause concentration and pH changes at the gel‐sample droplet interfaces and it is explained how these changes are related to ionic concentrations in the gel. The high concentration zone generated at one of the interfaces will be transported into the gel. This transport has been numerically simulated and experimentally verified. The low concentration generated at the opposite interface will cause titration impeding sample entrance in the gel through this interface even when the gel contains ions other than H+ or OH− transported towards the interface. The described phenomena explain the dependence of lateral spreading, precipitation at the application site as well as streaking and smearing along sample lanes, on the type and concentration of low molecular weight ions originally present in the gel.
Isoelectric focusing of basic proteins: The problem of oxidation of cysteines
Authors: Altland Klaus , Becher Pia , Rossmann Ute , Bjellqvist Bengt 
Published: May 2005

AbstractIsoelectric focusing of human globin chains in polyacrylamide gels dried in the ambient atmosphere and rehydrated in the presence of 8 mol/L urea produces artefactual doublets of zones as a result of oxidation by the gel. This oxidation can be avoided in separations of short duration by adding a reducing agent (e. g. 2‐mercaptoethanol or dithiothreitol to the rehydration solution (Altland, K. and Rossmann, U., Electrophoresis 1985, 6, 314–325)). We now demonstrate that the observed zone doublets can be explained by assuming neutralization of the contribution of dissociated sulfhydryl group of cysteine to pI by partial and reversible formation of globin dimers held together by disulfide bridges. Long time separations, requiring e. g. more than 4 h at > 500 V/cm, in pH gradients exceeding pH 7.5, are accompanied by artefactual oxidation from both the atmosphere and the gel matrix. Oxidation from the atmosphere as well as the effect of carbon dioxide can be eliminated by overlayering the gel with paraffin oil. Oxidation from the gel matrix can only partially be inhibited by rehydration of gels in the presence of 2‐mercaptoethanol or dithiothreitol. Nearly complete protection against oxidation by the gel matrix was achieved by adding a permanent supply of 2‐ME to the gel or by adding DTT to the cathodic wick towards the end of the experiment. Alkylation with iodoacetamide or iodoacetic acid resulted in stable globin patterns, which, however, displayed additional artefactual zones. Our experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.
Analysis of human apolipoproteins C by isoelectric focusing in immobilized pH gradients
Authors: Haase Regina , Menke‐Möllers Irene , Oette Kurt 
Published: May 2005

AbstractApolipoproteins C are involved in many ways in the metabolism of plasma lipoproteins. Apolipoproteins C from the delipidated VLDL of 35 controls and 165 normo‐and hyperlipoproteinemic patients were analyzed by isoelectric focusing on an immobilized pH gradient, pH 4.0–5.0, with 7 M urea, which raised the apparent pH range to 4.8–5.7. This method is an improvement over conventional isoelectric focusing with carrier ampholytes with regard to both resolution and reproducibility. Due to the high resolution (0.1 pH units per cm) additional apolipoprotein C‐III bands: C‐III0 A1, C‐III0 A2, C‐III1 C and C‐III2 C (the designations A, anodic, and C, cathodic, refer to direction of migration on IEF in relation to the main band) are described for the first time. The possible artifactual nature of these protein bands could be excluded. Cleavage with neuraminidase and peptidases, immunological detection and/or two‐dimensional electrophoresis were used to obtain more information. The additional bands seem, in part, to be hydrolysis products of carboxypeptidase A (C‐III1 C, C‐III2 C). The appearance of C‐III1 C and C‐III2 C was dependent upon the serum triglyceride concentration. The percent distribution of C apolipoproteins in very low density lipoproteins (VLDL) from control serum agreed with previously published data. Apolipoproteins C can also be focused in immobilized pH gradients from VLDL and serum without delipidation.