bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anomalous conductivity zones in electrophoresis. III. Experimental tests of the theory
Authors: Spencer Michael , Kirk Jean M. 
Published: May 2005

AbstractAn assumption used in developing the basic theory, that ionic retardation factors are substantially independent of solute concentration, is tested and found valid. The results of electrophoresis through 15% polyacrylamide gels are found to be in agreement with the theory. Zones of altered concentration appear in the presence of spermine tetrahydrochloride, sodium phosphate buffer (pH 7.2) or Tris‐HCl (pH 7.0). The use of ionic retardation factors and transport numbers deduced from conductivity measurements leads to correct prediction of the sign of concentration change in each case. The direction and velocity of migration of a low‐concentration boundary can also be predicted, together with associated changes in pH. Further confirmation comes from a detailed analysis of published work on electrophoresis through sucrose gradients. The theoretical treatment is suitable for application to other systems (such as isoelectric focusing and isotachophoresis) where a gel is used as a stabilizing medium, and where effects of the kind discussed may produce unexpected distubances.
Analysis of protein patterns during differentiation using 2‐D electrophoresis and computer multidimensional classification
Authors: Tarroux Philippe 
Published: May 2005

AbstractWe describe the application of a general method of comparing and sorting the patterns of protein expression obtained from two‐dimensional (2‐D) electrophoresis of a mixture of proteins. The method involved the use of two multidimensional classification programs and was applied, first, to the comparison of the protein patterns expressed at different stages during nymphal differentiation of imaginal wing discs in the insect Pieris brassicae. Comparison of these stages was based on the similarity of protein expression. The technique used allowed processing of all the information visible on a gel and took into account the variations in most of the spots labeled. Two qualitatively different stages of protein expression were found during the nymphal stage, and fine variations not revealed by any other method were shown to exist in genetic programing. Next, the method was applied to sorting the different polypeptides expressed; this was also done on the basis of their similarity of expression. We were able to characterize three groups of related peptides with common regulation features and apparently common physicochemical characteristics. The complete map of the most highly correlated peptides, which may constitute subunits of the same protein, was constructed by the programs, thus permitting fast rational analysis of protein expression. The clinical applications of these classification techniques are discussed.
Two‐dimensional electrophoretic analysis of myosin light chain phosphorylation in heart
Authors: Bárány Kate , Sayers Scott T. , Disalvo Joseph , Bárány Michael 
Published: May 2005

AbstractThe phosphorylatable light chain of myosin (P‐light chain) from hearts of the turtle, rat, frog, dog, cat, adult chicken and 1 week old chick was compared by two dimensional gel electrophoresis. The technique separates the phospho and dephospho forms of P‐light chain and allows quantitation of its percentage distribution. The P‐light chain was found to exist in multiple forms. The turtle heart possessed the highest percentage, 75%, of the phospho form of P‐light chain, whereas 25% of P‐light chain was in the dephospho form. In rat heart, 35% of the P‐light chain was phosphorylated and 65% dephosphorylated. In frog heart, the P‐light chain exhibited three forms: two phosphorylated and one dephosphorylated. The P‐light chain of dog, cat and adult chicken hearts displayed four spots on two‐dimensional gel electrophoresis: two phospho and two dephospho forms. In these species the percentage of total P‐light chain being in the phospho forms was in the range of 10–25%. Comparison of adult chicken and 1 week old chick preparations suggests that during maturation from the chick to the chicken the percentage phospho form of the total P‐light chain decreases. The myosin light chain kinase and phosphatase activities in whole homogenates of the above heart preparations were assayed. The data, with the exception of those from frog heart, suggest that the extent of light chain phosphorylation is correlated with the ratio of light chain kinase to phosphatase activity.
Improved procedures for controlling the voltage gradient and temperature during electrophoresis in submerged horizontal slab gels
Authors: Serwer Philip 
Published: May 2005

AbstractThe accuracy of electrophoretic mobility (μ) determinations made by electrophoresis in gels is limited by the accuracy of the control of voltage gradient and temperature. To improve the accuracy of μ determinations during electrophoresis in horizontal slab gels, apparatuses have been built and procedures have been developed for improved control of the voltage gradient and temperature during electrophoresis. Temperature is controlled ± 0.2–0.3°C and voltage gradient is controlled ± 1–2%.
Analysis of fetal peptides in human amniotic fluid and in fetal fluids and tissues by two‐dimensional electrophoresis
Authors: Jones Miren I. , Spragg S. Peter 
Published: May 2005

AbstractThe majority of the protein in mid‐trimester amniotic fluids is of maternal serum origin. However, analysis of aminiotic fluid by high resolution two‐dimensional electrophoresis revealed several proteins of non‐serum origin. The maps prepared from amniotic fluid were compared with those from various possible sources of the proteins, including fetal serum, cerebrospinal fluid and urine, and extracts of placenta, fetal kidney and lung. Several of the major constituents of fetal urine were identified in amniotic fluid, but the other fetal sources of proteins investigated here did not make a significant contribution to the non‐serum proteins in normal amniotic fluid. There appears to be a fetal modification of the α1‐antitrypsin in fetal serum as the pattern of this protein is different to that observed in maternal serum and in amniotic fluid.
Numerical measures of two‐dimensional gel resolution and positional reproducibility
Authors: Taylor John , Anderson N. Leigh , Anderson Norman G. 
Published: May 2005

AbstractProcedures to measure two important properties of two‐dimensional electrophoresis systems have been developed. These procedures evaluate the overall resolution and positional reproducibility of two‐dimensional gel patterns. Using these measures we show that various state of the art gel systems can produce patterns exhibiting resolution measures of 15 000–50 000 (yielding practical upper bounds of about 2000–6000 detectable proteins in typical cellular samples) and that positional accuracy for most spots in gel‐to‐gel comparisons is better than a spot width (0.5–2.0 mm). Implemented as part of a computerized image analysis system for two‐dimensional gel data, these two measures can be used in the selection and optimization of gel systems, and in the selection of sets of well‐resolved, well‐matched spots for genetic and other quantitative sudies.
Two‐dimensional gel electrophoresis of myelin and oligodendroglial proteins solubilized by a mixture of tetramethylurea and dimethylethyleneurea
Authors: Althaus Hans‐Henner , Klöuppner Sabine , Poehling Hans‐Michael , Neuhoff Volker 
Published: May 2005

AbstractA method is introduced which allows the solubilization of membrane proteins for isoelectric focusing without prior use of sodium dodecyl sulfate. Desalted and lyophilized samples of myelin and oligodendroglial membrane proteins are readily dissolved in a mixture of tetramethylurea and dimethylethyleneurea containing lysis medium. Subsequent isoelectric focusing in polyacrylamide gels reveals that the major portion of the myelin proteins focus at a rather alkaline pH. At least 6 components of the myelin basic protein can be distinguished, demonstrating its heterogeneity. Two‐dimensional gel electrophoresis reveals that the protein composition of myelin appears to be more complex than previously stated.
Fast and high resolution enzyme visualization in ultrathinlayer isoelectric focusing
Authors: Kinzkofer Angelika , Radola Bertold J. 
Published: May 2005

AbstractThree approaches are described for fast and high resolution enzyme visualization in ultrathin‐layer isoelectric focusing. (i) For direct visualization in the focused gel, solutions with reagent concentrations, increased 10–20 times over those normally used, were applied, accelerating and intensifying color development. Lactate dehydrogenase and alcohol dehydrogenase were visualized by overlayering the focused gel with the substrate and reagent solution. (ii) Membrane printing, employing polyamide or cellulose acetate membranes, pretreated with buffer and impregnated with the staining solution, was superior to direct visualization for some enzymes. Glycosidases were located by salting‐out in the focused gel with 90% ammonium sulfate, followed by printing with membranes impregnated with 4‐methylumbelliferyl derivatives of carbohydrates. (iii) Ultrathin (100–200 μm) agarose gels, equilibrated before use with solutions containing either low or high molecular weight substrates, buffers and other reagents, replace the traditional overlay technique. Proteases (Pronase P) and phosphoglucomutase were visualized with ultrathin replicas. With all approaches visualization was completed within 2–5 min. For some enzymes elevated temperatures in the range of 60–80°C accelerated visualization and drying of the gel. With all techniques a permanent document was obtained which could be evaluated densitometrically and conveniently stored for later reference. The combination of high resolution separation and detection, capable of handling up to hundreds of samples within a fraction of an hour, and a minimum of effort and costs, is an attractive option for many potential applications.
Quantification of a transferrin variant after isoelectric focusing in agarose gel using carbamylated myoglobin as a colored marker
Authors: Petrén Sven , Vesterberg Olof 
Published: May 2005

AbstractA widely applicable procedure for electrophoretic separation and quantification of proteins is described. Different colored markers of myoglobin were prepared by carbamylation and those with suitable isoelectric points (pI's) were isolated. Such markers were added to samples of serum and plasma which were examined by improved isoelectric focusing in agarose gel. This facilitated isolation of a certain transferrin with pI 5.7. This protein was quantified in gel pieces by zone immunoelectrophoresis assay. The overall S.D. was <10%. Immunofixations were performed to check the quality of focusing and the accuracy of gel punching.
The immunodetection of brain proteins blotted onto nitrocellulose from fixed and stained two‐dimensional polyacrylamide gels
Authors: Jackson Peter , Thompson R. J. 
Published: May 2005

AbstractProteins visualised by Coomassie Blue staining of two‐dimensional polyacrylamide gels of complex mixtures of brain proteins were blotted onto nitrocellulose sheets. The Coomassie Blue stain was transferred simultaneously and bound strongly to the nitrocellulose, giving a copy of the original gel pattern. Using specific antisera and a second antibody coupled to horseradish peroxidase, proteins in the original mixture could be detected on the nitrocellulose in quantities less than 25 ng. The Coomassie Blue and horseradish peroxidase stains were distinguishable by a suitable filter, allowing precise correlation of spots stained by Coomassie Blue with spots stained with the brown immunoperoxidase reaction product. The technique was used to identify related proteins in electrophoretograms of brain extracts from different animals.
Preparation of immobilized pH gradients spanning 2–6 pH units with two‐chamber mixers: Evaluation of two experimental approaches
Authors: Gianazza Elisabetta , Celentano Fabrizio , Dossi Giulio , Bjellqvist Bengt , Righetti Pier Giorgio 
Published: May 2005

AbstractAlternative procedures for the preparation of wide‐range immobilized pH gradients with two‐chamber mixers are compared. In the first approach, both vessels contain the same mixture of buffering species, with the addition of either a strong acidic or a strong basic titrant; with the second protocol, the concentration of the buffers is different in the two chambers. The formulations for twenty 2–6 pH unit‐wide ranges were computed according to both methods. Relevant parameters of the resulting gradients are listed, including deviation from pH linearity, buffering power, ionic strength and efficiency. The effect of mistakes in the preparation of the gradients is also discussed. “Different concentration” gels allow for a better linearity of the pH course; “same concentration” gels offer a more even buffering power and ionic strength, and assure better reproducibility, in consequence of both the reduced number of manipulations and their higher resistance to mistakes. Reagent consumption and efficiency are very similar in both cases.
High resolution of alpha‐1‐antitrypsin PI M subtypes by isoelectric focusing with an modified immobilized pH gradient
Authors: Weidinger Sebastian , Cleve Hartwig 
Published: May 2005

AbstractThe genetic varients of the human protease inhibitor system were examined by isoelecric focusing with a modified immobilized pH gradient. Classifications of PI varietns and PI M subtypes were made in the so‐called PI m8 zone. The common and rare inherited PI M subtypes could be readily distinguished.
Two‐dimensional electrophoresis of cerebrospinal fluid proteins in multiple sclerosis and various neurological diseases
Authors: Harrington Michael G. , Merril Cari R. , Goldman David , Xu Xian‐Hau , McFarlin Dale E. 
Published: May 2005

AbstractProteins from cerebrospinal fluid (CSF) were separated by two‐dimensional electrophoresis (2‐DE), visualized with silver staining and quantitated with computer‐assisted densitometry. This report describes the polypeptide variations observed and their correlations to disease states. CSF from 22 multiple sclerosis (MS) patients was compared to CSF from: 20 non‐inflammatory neurological disease cases (NINDC), 17 inflammatory neurological disease cases (INDC) including a sub‐set of 6 patients with sub‐acute sclerosing panencephalitis (SSPE) and 14 normal volunteers. In a specific comparison between the MS group and the normal volunteers, 32% of the polypeptides varied quantitatively (p < 0.05 or greater). The NINDC group resembled the normal volunteers, while the INDC and SSPE groups resembled the MS group. In general, the quantitative changes in the polypeptides analysed in this study permit one to distinguish one disease state from another. In addition to these quantitative alterations, we also report the appearance of additional immunoglobulin light chain species in MS, SSPE, Herpes simplex encephalitis, neurosyphilis, one patient with Shy‐Drager syndrome and one patient with idiopathic orthostatic hypotension. No other disease‐specific qualitative changes were observed in the form of either deletions of proteins normally present, or additional proteins. This survey utilizing 2‐DE has demonstrated protein alterations in the CSF of patients with varied neurological diseases compared with normal CSF, which may prove useful in clinical diagnosis.
Silver staining of human salivary proteins following two‐dimensional electrophoresis using either protein denaturing or non‐denaturing conditions
Authors: Marshall Thomas 
Published: May 2005

AbstractThe proteins of human whole saliva have been detected by either silver or Coomassie Brilliant Blue R‐250 staining following two‐dimensional electrophoresis using either protein denaturing or non‐denaturing conditions. The high resolution protein denaturing technique revealed over 600 polypeptides and the non‐denaturing technique over 200 proteins in 50 μl of unconcentrated saliva. Silver staining enhances (50–100 fold) detection of the majority of these salivary components and reveals (as clear spots) both polypeptide and proteins not detected with Coomassie Brilliant Blue R‐250. Nevertheless, optimal silver staining of proteins (non‐denaturing conditions) requires pre‐equilibration of the gels in aqueous sodium dodecyl sulphate. In contrast, Coomassie Brilliant Blue R‐250 reveals diffusely staining pink zones not detected by silver staining. The possible nature of salivary components demonstrating unusual staining characteristics is discussed.
Improved procedure for silanization of glass plates as supports for polyacrylamide gels
Authors: Neuhoff Volker 
Published: May 2005

AbstractSilanization of plastic foils or glass plates is necessary for fixation of polyacrylamide gels to the support. When using glass plates as support, silanization creates problems which can be completely avoided when the silanized plates are heated for 30–45 min at 55–60°C prior to gel polymerization.
Sensitive silver staining of proteins after isoelectric focusing in agarose gels
Authors: Vesterberg Olof , Gramstrup‐Christensen Birte 
Published: May 2005

AbstractAs isoelectric focusing in agarose gels has many merits it is valuable to be able to visualize proteins at concentrations lower than detectable with Coomassie Brilliant Blue R‐250 staining. A silver staining procedure has been adapted to such gels and many proteins including serum samples have been studied. Silver staining gave very sharp protein bands and was about 30 times more sensitive than Coomassie Brilliant Blue R‐250 staining. This is important to allow direct application of dilute protein solutions such as urine and cerebrospinal fluid. This may also avoid artifactse.g.caused by concentration methods.
Typing of esterase D by isoelectric focusing
Authors: Budowle Bruce 
Published: May 2005

AbstractA method is described for obtaining reproducible resolution of Esterase D (EsD) phenotypes from human bloodstains by ultrathin‐layer polyacrylamide gel isoelectric focusing. By employing a pH 4.5–5.5 gradient and an electrode wick distance of 9.5 cm, all the common EsD variants were separated in 85 min. Electrofocusing of EsD took 53 and 60 min on the Cold Focus Apparatus and Ultrophor, respectively, when the distance separating the electrodes was 8.0 cm. Bloodstains up to 4 weeks old could be readily typed for EsD using this method.
The use of silver stain in sodium dodecyl sulfate polyacrylamide gel electrophoresis of immunoprecipitates obtained by crossed immunoelectrophoresis and liquid precipitation
Authors: Cutts J. Lynn , Sweet George H. , Tegtmeier Gary E. 
Published: May 2005

AbstractSilver staining was examined as a means for detecting antigenic polypeptides separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) after recovery from immunoprecipitates produced by crossed immunoelectrophoresis and liquid precipitation. Both precipitin procedures resulted in detection of the expected polypeptides with bovine serum albumin (BSA) and human transferrin as model antigens, as well as microheterogeneity of BSA, whereas antigenic polypeptides were rarely detected with Coomassie Brilliant Blue staining of SDS‐PAGE. The technique is advantageous over the use of radiolabeled antigens in being less expensive and time consuming and over antibody‐blot procedures in detecting antigenic activity prior to the denaturing conditions of SDS‐PAGE. Possible drawbacks are the appearance of antibody polypeptide chains on the electrophorograms as well as other non‐antigenic components when crude extracts of virally‐infected cells are used as antigen.
Wedge‐Shaped ultrathin polyacrylamide and agarose gels for isoelectric focusing: A new method for typing phosphoglucomutase (PGM1) in semen stains and vaginal swabs
Authors: Pflug Werner 
Published: May 2005

AbstractA modified method of ultrathin‐layer isoelectric focusing is described. The method allows subtyping of phosphoglucomutase even in samples containing considerable amounts of salts and proteins without pretreatment. Wedge‐shaped polyacrylamide or agarose gels varying in thickness from 50 μm (cathode) to 300 μm (anode) are used. If the right pH gradient is chosen the isoenzymes will focus near the cathode where the gel is ultrathin. The sample is applied anodally where the gel is thickest and the capacity therefore high. Thus the effect of salts and proteins on the pH gradient is diminished. Comparative tests have shown that this method is superior to the conventional techniques using gels with a uniform thickness of 100 μm.
Quantitation of nanogram amounts of glycosaminoglycans in samples containing proteins by a new densitometric method
Authors: Bertolotto Antonio , Magrassi Maria L. 
Published: May 2005

AbstractGlycosaminoglycans, separated electrophoretically and stained with Alcian Blue 8GX, were quantified by densitometry. The method allows quantitation of all classes of glycosaminoglycans in samples containing up to 10 mg/ml of serum proteins. Detection sensitivity is 40–300 ng and the optimum range of analysis is either 40–400 or 300–2100 ng, depending on the classes of glycosaminoglycans examined. The method is based on the densitometric comparison between a reference standard of 150 ng of hyaluronate and biological samples electrophoresed on the same sheet. By densitometry the total glycosaminoglycan concentration as well as the percentage and concentration of single fractions are determined. Each class of glycosaminoglycans presents different affinity to Alcian Blue, resulting in over‐ or underestimation in comparison with hyaluronate. Such error is constant over the whole range of determination and may be corrected by correction factors evaluated for each class. Both electrophoresis and densitometric quantitation may be optimized for an analysis of different glycosaminoglycans extracted from biological samples. For instance, heparin and heparan sulfate may be identified by nitrosation applied directly on the sheet; each class may be quantified by comparison with its own standard glycosaminoglycan, avoiding over‐ or underestimation; moreover, dermatan sulfate may be subdivided into fractions. The method is suitable for the quantitation of glycosaminoglycans present in low concentration in biological samples.
Transferrin and plasminogen phenotyping of aging blood stains using isoelectric focusing and silver stain
Authors: Dykes Dale D. , Miller Shirley A. , Polesky Herbert F. 
Published: May 2005

AbstractA three‐month study was conducted on blood stains to determine the reliability and viability of the transferrin (Tf) and plasminogen (PLG) genetic markers upon storage under varying conditions. Stains stored at room temperature (19–22 °C), 4 °C and −70 °C were tested at weekly intervals. Isoelectric focusing on agarose gels was followed by fixation and staining with Coomassie Brilliant Blue R‐250. Gels were subsequently treated with silver stain if band intensity was too faint for phenotyping. Stains stored at room temperature and 4 °C showed a gradual decrease in band intensity. By using silver stain, it was possible to phneotype blood stains stored for at least twice the length of time as compared to those stained with Coomassie Blue. This study demonstrates that isoelectric focusing and silver staining is reliable for determining Tf and PLG markers in forensic investigations.