bluuebunny/crossref_metadata_2025_split huggingface dataset:
Experimental evidence for flexible slope of immobilized pH gradients poured under computer control
Authors: Altland Klaus , Hackler Rolf , Banzhoff Angelika , von Eckardstein Arnold 
Published: May 2005

AbstractRecently, Altland and Altland (Clin. Chem.1984,30, 2098–2103) described three alternative devices for pouring immobilized pH gradients (IPGs) based on simultaneous delivery of a density gradient and a non‐density gradient by using high precision burettes under computer control. One of the expected improvements was an unlimited flexibility for the slope of the non‐density gradient (e. g.the pH gradient) which is stabilized by the density gradient. The human plasma proteins prealbumin and apolipoprotein AI and some of their inherited variants are demonstrated by double one‐dimensional electrophoresis in the sequence polyacrylamide gel electrophoresis followed by isoelectric focusing (IEF) in IPGs of varying slopes in the range of pH 4 to 7. It is shown that the patterns obtained follow the expectations from the gradients drawn on the computer. We conclude that the new devices for pouring IPGs can provide a valuable tool for the easy handling of complex separation problems to be solved by IEF.
Heterogeneity of factor XIIIB: A new method for the determination of factor XIIIB phenotypes by isoelectric focusing in 6 M urea
Authors: Kamboh Mohammad I. 
Published: May 2005

AbstractPreviously reported anomalous results obtained on the separation of the factor XIIIB (F XIIIB*2) allele product have been reinvestigated by thinlayer polyacrylamide gel isoelectric focusing (IEF). The ordinary IEF fails to resolve the difference between the products of F XIIIB*1 and F XIIIB*2 alleles. However, IEF in polyacrylamide gel in the presence of 6 M urea has been found to be a useful method of differentiation between these two allele products.
Automated, rapid image analysis and densitometry for enzyme linked immunosorbent assay, dot‐blot, and two‐dimensional gels using a microcomputer and electrooptical device
Authors: Lockshin Richard A. 
Published: May 2005

AbstractA commercially available electrooptical device, coupled to a microcomputer, is capable of analyzing densitometric values with a resolution of 280 × 128 points (35 840 individual points). A complex autoradiogram of a two‐dimensional electrophoretic gel is completely sorted into X and Y coordinates and exposure values differeing by as little as 0.01 O.D. (optical density) units or over an O.D. range of 0 to 4.00 in less than 2 min. For other purposes such as comparison of two gels, immunoelectrophoretic patterns, enzyme linked immunosorbent assay or microtitration plates, dot‐blot assays, or tissue samples, a comparison may be made and all differences stored in the computer in less than one second and further decisions or computer‐driven actions decided on that basis.
Isoelectric focusing of unconcentrated cerebrospinal fluid: Applications to ultrasensitive analysis of oligoclonal immunoglobulin G
Authors: Staugaitis Susan M. , Shapshak Paul , Tourtellotte Wallace W. , Lee Mike M. , Reiber Hans‐Otto O. 
Published: May 2005

AbstractThe application of isoelectric focusing (IEF), immunoglobulin G (IgG) immunofixation and silver staining for the analyses of unconcentrated cerebrospinal fluids (CSF) and sera are described. We have standardized these methods, and applied them to IgG oligoclonal band analysis. The sensitivity of the procedure was 0.3 μg IgG. Optimum results were obtained with 0.5 μg IgG in 5–20 μl application volumes. Equal quantities of CSF and serum IgG analyzed by IEF in 55 normal individuals showed comparable intensities and isoelectric points and of these, four individuals (8%) had bands in CSF which were not detectable in serum. The CSF and sera from patients with multiple sclerosis, subacute sclerosing panencephalitis, Huntington's disease and Guillain‐Barré syndrome were also examined. Pathologic banding patterns can be classified into three categories: Bands present (1) exclusively in CSF, (2) more intense in CSF compared to serum, and (3) equally intense in CSF and serum. Oligoclonal IgG bands are defined as bands found exclusively or more intense in CSF compared to serum, and are indicative of intra‐blood‐brain‐barrier IgG synthesis.
Fabric reinforced polyacrylamide gels for electroblotting
Authors: Nishizawa Hideyuki , Murakami Ayako , Hayashi Naoko , Iida Mami , Abe Yoshihiro 
Published: May 2005

AbstractThe preparation of 0.5 mm fabric reinforced polyacrylamide (FRP) gels is described. After isoelectric focusing, egg white and isoelectric point marker proteins were quantitatively transferred in only 20 min from the FRP gels to nitrocellulose membranes by electroblotting. FRP gels combine the advantages of previously described supports for polyacrylamide gels with penetrability for electric current during blotting. The gels may be conveniently stored and afford operational versatility.
A fluorescent method for the rapid staining and quantitation of proteins in sodium dodecyl sulfate‐polyacrylamide gels
Authors: Aragay Anna M. , Diaz Pilar , Daban Joan‐Ramon 
Published: May 2005

AbstractFluorescence studies carried out in solution indicate that in the presence of the anionic detergent sodium dodecyl sulfate (SDS) proteins are able to bind the noncovalent dye 1‐anilinonaphthalene‐8‐sulfonate (ANS). The interaction of this hydrophobic dye with protein‐SDS complexes is probably responsible for the fluorescence of protein bands observed by UV‐transillumination of polyacrylamide gels stained with ANS. It is shown that the staining of SDS‐polyacrylamide gels with ANS after electrophoresis allows the detection and quantitative estimation of proteins and peptides having different properties. The electrophoretic patterns obtained using ANS are equivalent to those obtained using Coomassie Brilliant Blue R‐250. The fluorescent staining method described in this work is simpler and much more rapid than conventional methods using visible dyes.
Thin‐layer counter current line absorption immuno‐electrophoretic analysis of antigens and antibodies to aleutian disease virus ‐ a mink parvovirus
Authors: Alexandersen Søren , Hau Jann , Aasted Bent , Poulsen Otto M. 
Published: May 2005

AbstractA thin‐layer counter current line absorption immunoelectrophoresis (TL‐CCLAIE) assay was developed for the analysis of antigens and antibodies to Aleutian disease virus. The TL‐CCLAIE assay is a rapid and sensitive assay employing 0.4 mm gels cast in moulds in order to allow technicians not routinely engaged in electrophoretic work to perform the assay. Thin gels allow a higher voltage and thus higher electrophoretic mobility of the antigens, making it possible to perform the complete assay within 2 h.
The formation of stable pH gradients with weak monovalent buffers for isoelectric focusing in free solution
Authors: Mosher Richard A. , Thormann Wolfgang , Graham Aly , Bier Milan 
Published: May 2005

AbstractTwo methods which utilize simple buffers for the generation of stable pH gradients useful for preparative isoelectric focusing are compared and contrasted. The first employs preformed gradients comprised of two simple buffers in density‐stabilized free solution. The stability of this system is analyzed theoretically and by computer simulation. These precast gradients are limited to two buffering components, subject to diffusion, and restricted to the neutral pH region. An experimental application is presented. The second method utilizes neutral membranes to isolate electrolyte reservoirs of constant composition from the separation column. It is shown by computer simulation that steady state gradients can be formed at any pH range with any number of components in such a system.
Quantitation of microgram amounts of protein in two‐dimensional polyacrylamide gel electrophoresis sample buffer
Authors: Ramagli Louis S. , Rodriguez Lewis V. 
Published: May 2005

AbstractThe concentration of protein actually solubilized in sample buffer in preparation for analysis by two‐dimensional polyacrylamide gel electrophoresis cannot be directly determined by the Lowry, Biuret, or Bradford protein methods due to interference by the combinational effect of presence of urea, detergents, carrier ampholytes, and thiol compounds in sample solubilization buffers. Determinations of the actual amount of protein solubilized and applied to gels is required to accurately quantitatively and qualitatively evaluate second‐dimension polypeptide maps. It was found that when sample buffer consisting of 9 M urea, 4 % Nonidet P‐40, 2 % Ampholines, and 2 % 2‐mercaptoethanol containing solubilized sample(s) was acidified prior to dilution, protein concentrations over a range of 0.5 to 50 μg could be reproducibly determined utilizing a modified Bradford assay. The modified assay generates two near‐linear segments, one over the range < 0.5 to 5 μg total protein that permits the application of Beer's law and a second linear response encompassing 5 to 50 μg total protein. The assay did not tolerate presence of greater than 0.1 % sodium dodecyl sulfate but addition of sodium chloride and protamine sulfate did not adversely affect protein quantitation. The modified assay allows direct quantitation of protein solubilized in sample buffers containing urea, carrier ampholytes, nonionic detergents, and thiol compounds.
Effects of aroclor 1254 on proteins of mouse liver: Application of two‐dimensional electrophoretic protein mapping
Authors: Leigh Anderson N. , Swanson Mark , Giere Frederic A. , Tollaksen Sandra , Gemmell Anne , Nance Sharron , Anderson Norman G. 
Published: May 2005

AbstractLiver proteins of male C57BL/6T mice treated with 0, 50, or 250 mg/kg Aroclor 1254 were analyzed by high‐resolution two‐dimensional (2‐D) electrophoresis. The resulting patterns were processed using a computerized image analysis system and quantitative data selected for a total of 150 protein spots. On the basis of an analysis of liver proteins form five animals in each treatment group, we found 31 proteins that showed quantitative differences attributable to treatment with chlorinated hydrocarbons at a high level of statistical significance. One of the altered proteins appears to be Mitcon:2, a heat‐shock sensitive mitochondrial matrix polypeptide; another appears likely to be microsomal cytochrome b5. The results indicate that quantitative 2‐D protein mapping may reveal much more detail regarding the in vivo effects of toxic xenobiotics than has previously been available, and thus allow a more informative approach to the testing of toxic compounds.
Technical improvements in two‐dimensional electrophoresis increase the level of genetic variation detected in wheat‐seedling proteins
Authors: Damerval Catherine , De Vienne Dominique , Zivy Michel , Thiellement Hervé 
Published: May 2005

AbstractThe level of genetic variation revealed by two‐dimensional electrophoresis of proteins from seedlings of two wheat lines strongly depends on the technical procedures. Improvements in extraction and electrophoresis procedures relative to earlier experiments on the same material led to a significant increase in the genetic variation revealed: 15.2 % instead of 6.7 % of the spots were genetically variable. The improved procedure is based on (i) precipipation of proteins from wheat seedlings with trichloroacetic acid and acetone, (ii) solubilization of the proteins with a solution containing urea, potassium carbonate and sodium dodecyl sulfate, (iii) isoelectric focusing in an optimized pH gradient, obtained with a mixture of carrier ampholytes (Pharmalyte and Servalyt), and (iv) running elecrophoresis in the second dimension on gels with increased surface.
Improved design of one‐dimensional vertical polyacrylamide slab gel electrophoresis avoids lateral streaking
Authors: Neuhoff Volker , Cheong‐Kim Kyong‐Sun , Altland Klaus 
Published: May 2005

AbstractLateral streaking or bending in vertical one‐dimensional slab gel electrophoresis can be completely avoided if a comb‐gel made up in electrophoresis buffer is placed on top of the stacking gel or sample gel.
Purification of human myoglobin by preparative flat bed isoelectric focusing
Authors: Samaan Ghassan , Caudie Christiane , Quincy Claude 
Published: May 2005

AbstractWe have prepared a standard of human myoglobin by flat bed isoelectric focusing, to be used in immunological and chemical methods of measurement in clinical biology. Myoglobin was extracted from human psoas or myocardium and then purified by preparative isoelectric focusing in a granulated gel. The purified myoglobin was examined by analytical isoelectric focusing, high performance liquid chromatography (HPLC), immunoelectrophoresis, and two‐dimensional gel electrophoresis. Myoglobin was quantified by three methods: (i) Drabkin's method, and by the determination of iron using (ii) the bathophenanthroline method, and (iii) the atomic emission method. There was good correlation between all three methods. Purified myoglobin was stabilized by conversion to the cyanmetmyglobin form, and stored until use at 4 °C (± 2 °C). or at – 20 °C in the liquid state after addition of ethylene glycol.
On the reproducibility of band position in electrophoretic separations
Authors: Gianazza Elisabetta , Astrua‐Testori Silvia , Caccia Paolo , Giacon Patrizia , Quaglia Laura , Righetti Pier Giorgio 
Published: May 2005

AbstractThe reproducibility of protein migration was studied in three electrophoretic systems: sodium dodecyl‐sulfate electrophoresis on polyacrylamide gradient, isoelectric focusing on immobilized pH gradients and two‐dimensional charge‐size separations. The gradient gels were poured either with a two‐vessel apparatus or with a set of computer‐driven burettes, as described by Altland and Altland (Electrophoresis 1984, 5, 143–147). Standard deviation on band position was cut by 20–50% with the latter approach in comparison with the former. Typical results were: sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, 0.6 mm (over a total length of 135 mm); immobilized pH gradients, 0.6 mm (for 179 mm length); two‐dimensional separations, 0.8 mm (χ‐axis), 0.6 mm (y‐axis).
Subtyping group‐specific component or esterase D using the same ultrathin‐layer polyacrylamide gel format
Authors: Budowle Bruce 
Published: May 2005

AbstractAn ultrathin‐layer polyacrylamide isoelectric focusing gel is described for obtaining highly resolved, nondistorted, reproducible patterns for group‐specific component (Gc) or esterase D (EsD) subtyping. The 1F, 1S, 2 and 1A1 bands of the Gc system were all typeable by isoelectric focusing alone. All common EsD allelic products were easily resolved. Typing Gc or EsD derived from bloodstains was also demonstrated.
A micromethod for the preparation of tissue from defined fractions of a Drosophila head and its analysis by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Müller Uli , Spatz Hans‐Christof 
Published: May 2005

AbstractA method is described which allows the preparation of retinae, laminae, opticallobes, protocerebrum and proboscis of single heads of Drosophila. The protein content of these tissues was examined with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue R‐250 and glycoprotein staining. A catalog of 39 major proteins or protein subunits was obtained. Their relative proportions were determined quantitatively for the different tissues from Drosophila wildtype strains AS and C‐S as well as the mutant sevLY3.
A new applicator strip with a concentration effect for isoelectric focusing
Authors: Pflug Werner , Laczko Bela , Müller Fritz 
Published: May 2005

AbstractThe sensitivity for protein detection after isoelectric focusing can be enhanced using a newly developed applicator strip with a concentration effect. This effect is achieved with funnel‐shaped slots, which are placed parallel to the pH gradient. Furthermore, sample volumes varying from 1–90 μL can be applied without distortion to the pH gradient. A comparison with other commercially available applicator strips was undertaken using the phosphoglucomutase system.
Analysis of convex Ferguson plots in agarose gel electrophoresis by empirical computer modeling
Authors: Tietz Dietmar , Chrambach Andreas 
Published: May 2005

AbstractAgarose gel electrophoresis has been shown to give rise to non‐linear plots of log (mobility) vs. gel concentration of spherical viruses (Serwer,[1]) and cellular vesicles (Gottlieb et al.[2]). This finding also applies to proteins as shown in this study. Considering that in the non‐linear plot, the slope becomes a function of gel concentration, it is possible to determine particle properties and gel parameters by a modification of the conventional method derived from the Ogston theory for long‐fiber gels. This treatment shows: (a) In application to data obtained from gel electrophoresis (0.4 to 1.6 % agarose) of viruses (13 to 42 nm radius) and with increasing gel concentration: (i) an increase of apparent total fiber length per g agarose matrix, (ii) a reduction of apparent fiber radius and (iii) a constant fiber volume (per g matrix material) of the agarose fiber. The values of the fiber radii identify the fibers as agarose supercoils (or aggregates of it) with a radius of 20–55 nm. (b) In application to data obtained from gel electrophoresis (1.2 to 8 % agarose) of proteins (1.7 to 5.8 nm radius): a fiber volume indicative of additional sieving by the agarose double helix (of known radius of 0.5–2 nm). This is in agreement with a previous suggestion by Serwer [1] that proteins are able to penetrate into the double‐helical network of which the agarose supercoil consists. (c) The likelihood of a continuous transition from case (a) to case (b).
Avoiding liquid exudation on the surface of rehydrated gels used for hybrid isoelectric focusing in carrier ampholyte supplemented immobilized pH gradients
Authors: Altland Klaus , Hackler Rolf , Rossmann Ute 
Published: May 2005

AbstractRecently, we described hybrid isoelectric focusing in rehydrated polyacrylamide gels as an analytical procedure which is based on separation in immobilized pH gradients supplemented with low concentrations of carrier ampholytes (Altland and Rossmann, Electrophoresis 1985, 6, 314–325). When using this procedure at various pH ranges, in the presence of reducing agents and carrier ampholytes at different concentrations and from different suppliers, over prolonged time intervals for separation and at various acrylamide concentrations in the gel, liquid exudation has been observed on the gel surface which may result in a confluent liquid layer adversely affecting the final separation pattern. The effect is demonstrated and procedures are described to overcome the phenomenon by adding polyols like glycerol, sorbitol, sucrose or dextrans to the rehydration solution. Comparable effects are achieved when these components are added at the same concentration by weight rather than by moles. The addition of 10 g % Dextran 8 or 20 g % sucrose or sorbitol to the rehydration solution prevents liquid exudation for several hours. These additives are compatible with high concentrations of urea, with reducing agents and with neutral detergent (Triton X‐100).
A technical note on esterase D subtyping
Authors: Rocha Jorge , Amorim AntóNio 
Published: May 2005

AbstractThe electrophoretic behavior of esterase D (E.C. 3.1.1.1.) allozymes 1, 2 and 5 was studied in various buffers and pH ranges, and compared with results obtained on isoelectric focusing. It is shown that the relative mobility of ESD 2 and 5 allozymes is reversed according to the pH of the buffer system. In acidic pH ranges the mobility is: cathode 1<2<5 anode; using the same buffer system in basic pH ranges the following order is observed: 1<5<2. Technical improvements in agarose gel electrophoresis leading to a considerable economical advantage over the previously described methods are presented.
On the relationship of amino acid composition to silver staining of proteins in electrphoresis gels
Authors: Gersten Douglas M. , Wolf Pamela H. , Ledley Robert S. , Rodriguez Lewis V. , Zapolski Edward J. 
Published: May 2005

AbstractDevelopment of techniques for silver staining of proteins separated by polyacrylamide gel electrophoresis has increased experimental detection limits. However, the precise basis for the reaction between silver and polypeptides is still unclear and, depending upon the choice of silver reagent, may even differ. We compared protein stain intensity with silver diamine (ammoniacal silver) and silver nitrate reagents based on amino acid composition by determining the protein amount required to generate an arbitrary staining intensity and then calculating moles of each amino acid present in some representative proteins. We compared these values for each amino acid, taken singly, as well as for combinations of two and more. In no case could staining be attributed entirely to specific or classes of amino acids, thus supporting the argument that an inherent difference exists between primary reactive centers for the two staining reagents.
Decrease in the mobility of proteins during electrophoresis in linear gradients of polyacrylamide
Authors: Lambin Patrick , Herance Nicole , Fine Jean‐Marius 
Published: May 2005

AbstractProteins with molecular weight between 66 000 and 950 000 were subjected to electrophoresis in 3 to 30 % linear gradients of polyacrylamide with volt‐hour products between 2000 and 20 000. From the experimental data it was concluded that displacement of proteins decreased proportionally to the logarithm of time of electrophoresis. With these coordinates the fitness of the curves was found to be excellent (correlation coefficients were better than 0.993 for the analyzed proteins). These results support the hypothesis that there is no limit to be reached by a protein during its migration in a linear gradient of polyacrylamide. In addition we observed that the rate of decrease in mobility was proportional to the size of the protein.
Human antibody‐secreting cells enriched by free flow electrophoresis
Authors: Bauer Johann , Hannig Kurt 
Published: May 2005

AbstractMononuclear leukocytes of the human peripheral blood were separated by counter‐current centrifugal elutriation. In this way a group of lymphocytes was isolated, which contained B‐lymphocytes with an electrophoretic mobility (EM) of 0.9 × 10−4 (cm2 V−1 s−1) and T‐lymphocytes with an EM of 1.1 × 10−4 (cm2 V−1 s−1). This group of lymphocytes was incubated for 7 days in the presence of monocytes and pokeweed mitogen. Then the culture supernatants were screened. Non‐specific human antibodies were found in all culture supernatants. In 20% of the supernatants human antibodies against sheep red blood cells could be detected. The viable cells of the cultures were separated into 5 fractions by free flow electrophoresis. Then the cells of the different fractions were tested for their capability to produce antibodies. The tests revealed that antibody‐secreting cells had an EM around 0.9 × 10−4 (cm2 V−1 s−1) and that the greater part of the cells, isolated from pokeweed mitogen cultures, had a significant higher EM. Thus free flow electrophoresis could be applied to enrich antibody‐secreting cells ninefold.