bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation of stable pH gradients for preparative isoelectric focusing by electrolysis of two‐component buffer solutions in a multi‐compartment apparatus
Authors: Jonsson Mats , Fredriksson Stig 
Published: May 2005

AbstractA widened use of isoelectric focusing for preparation of large amounts of pure proteins is highly desirable but is severely hampered,inter aliaby the high cost of the carrier ampholytes used for creating the pH gradient. Efforts are therefore being made to replace the carrier ampholytes with simple two‐component buffers. To this end, a multimembrane electrolyzer, consisting of 22 compartments with attached cooling loops, has been constructed. The choice of membranes has proved to be one of the main problems. With permeable membranes, even small differences in hydrostatic pressure between the ends of the electrolyzer will cause internal liquid flows that prohibit the formulation of a useful pH gradient. Likewise, flow‐tight membranes generally give rise to a large internal flow due to electroosmosis. Among the types of membranes so far examined, polycrylamide membranes form an exception. With these membranes, useful and stable pH gradients have been created. There are drawbacks, however, relating to the low mechanical strength and to the difficulty of producing exactly equal membranes, thereby fulfilling the theoretical demand of constant transference numbers for the ions. The experimental observation that even small disturbances, such as variations of the transference numbers or a minute electroosmotic flow through the electrolyzer, may completely destroy the pH gradient, has been related to the mechanism operating during electrolysis of a simple buffer. While a possible local breakdown of a carrier‐ampholyte pH gradient is rapidly repaired by the current, a disturbance occurring in a buffer pH gradient can be repaired only by diffusion, which is a slow process.
Polymerization kinetics of polyacrylamide gels I. Effect of different cross‐linkers
Authors: Gelfi Cecilia , Righetti Pier Giorgio 
Published: May 2005

AbstractPolymerization kinetics of polyacrylamide gels, cross‐linked with the following: N,N′‐methylenebisacrylamide (Bis), N,N′‐bisacrylylcystamine (BAC), N,N′‐ diallyltartardiamide (DATD), N,N′(1,2‐dihydroxyethylene) bisacrylamide (DHEBA) and ethylene diacrylate (EDA), have been studied. The gels are polymerized directly in a spectrophotometer quartz cuvette and the kinetics followed at 283 nm (disappearance of the double bond) or at 600 nm (Tyndall effect due to turbidity of Bis, DHEBA and BAC gels). The order of reactivity of the various cross‐links appears to be: Bis ≅ DHEBA > EDA ≅ BAC ≫ DATD. The last cross‐link, (DATD), was found to actually be an inhibitor of gel polymerization, leading to highly unpolymerized gels, especially at high %C values. Bis and DHEBA gels, at 3 to 5 %C levels, are fully polymerized within 30 min at room temperature, while EDA and BAC gels, in the same %C range, require at least 3h. All cross‐links, when used above 10 %C, display quite slow polymerization kinetics, requiring overnight reaction for good conversion of monomers into polymer chains. When polymerizing liquid linear polyacrylamide (without cross‐link), an initial concentration of > 5% should be used and the reaction continued overnight, at room temperature or higher, for acceptable polymerization efficiency. On the basis of these data, a new structural model for highly cross‐linked gels is proposed.
The application of direct tissue isoelectric focusing to the study of human skeletal muscle
Authors: Thompson Basil J. , Burghes Arthur H. M. , Dunn Michael J. , Dubowitz Victor 
Published: May 2005

AbstractA method of direct tissue isoelectric focusing (DTIF) in agarose of human skeletal muscle is described. This particular method was developed to utilize the small amounts of tissue obtained by needle muscle biopsies performed for diagnostic purposes. 20 μm thick cryostat sections were adhered to the hydrophilic surfaces of small GelBond rectangles. These were then applied directly to the surface of the gels. A charge‐balanced purified agarose was used to make the gels, which contained Triton X‐100 to enhance protein solubilization. Stabilization of the pH gradient was attempted by employing a 3% w/v ampholyte which was a blend of 0.85% w/v pH 8–10.4 and 2.15 % w/v pH 3–10 Pharmalyte, by using anolyte regulation with 0.1 M aspartic acid and by focusing the gels under a CO2‐extracted nitrogen atmosphere. Equilibrium of proteins stained by Coomassie Brilliant Blue R‐250 was apparent as monitored by comigration of sections from both anode and cathode. With the exception of the cathode end, excellent resolution and reproducibility was achieved. Better cathode resolution was noted with non‐equilibirium conditions. Preliminary zymograms of lactic dehydrogenase (LDH), using a tetrazolium technique, have shown consistent patterns of multiple isoenzymes, the basic components of which were best seen using non‐equlibirium conditions.
Steady‐state rheoelectrolysis — a method for sioelectric focusing without carrier ampholytes. II. The pH course to be expected on rheoelectrolysis of a buffer solution composed of a weak acid and a weak base
Authors: Rilbe Harry , Stockholm  
Published: May 2005

AbstractSteady‐state rheoelectrolysis of a salt of a weak base and a weak acid, with a possible excess of base or acid, is treated theoretically. For an accurate deduction of the pH course through the region where the pure salt prevails, the treatment is based directly on the mass action equations and not on Henderson's equation. However, the conductivity contributions of the water ions H+ and OH− are not taken into account, a limitation that restricts the validity of the theory roughly to the pH region between 4 and 10. The pH span that can be covered by this technique is found to be about 2.5 units bigger than the pK difference between base and acid. An exact expression for the pH gradient is deduced, and it is shown that this gradient has a maximum at the location of the pure salt and minima in the neighbourhood of the two pK values. There is no theroretical obstacle to the use of this technique for isoelectric focusing without carrier ampholytes.
Isozyme comparisons using weighted analyses of electrophoretic mobility in a range of polycrylamide gel concentrations
Authors: Fieldes Mary A. , Tyson Hugh 
Published: May 2005

AbstractPeroxidase isozymes from two flax genotypes were separated electrophoretically using a range of concentration, regression models were fitted by weighted least squares method, using matrix techniques and appropriate programmes, weight were reciprocals of variances of significance, as a chi square, of the residual sums of square (SS). Log (Rm) Plotted against gel concentration showed significant curvilinearity; Log (P/Hb), where P was an isozyme's migration was linear with concentration. Matrix method allowed a completely generalized approach to the analysis of data of this type. In particular, regression data could be readily combined so as to allow orthogonal comparison between Intercepts and slops in an analohgous fashion to the orthogonal breakdown of row, column and interaction effects in a balance two‐way analysis of Weighting effected some improvement in comparison between isozymes; combined analyses of weighted regression produced striking improvement in discrimination relative to simple pairwise Comparisons between isozymes.
Silver Stains for proteins in polyacerylamide gels: A comparison of six methods
Authors: Ochs Denis C. , McConkey Edwin H. , Sammons David W. 
Published: May 2005

AbstractSix of the most recent silver staining method for detecting polypeptides on polyacrylamide gels were compared. We found the method of Sammons et al. (Electrophoresis, 1981, 2, 135–141) to be least expensive and most reproducible, For staining gels of 1.5 mm in thickness, it is also the most sensitive method. Other methods may be preferable for staining gels 0.8 mm or less in thickness.
Determination of molecular weights and Stokes' radii of non‐denatured proteins by polyacrylamide gradient gel electrophoresis. 1. An equation relating total polymer concentration, the molecular weight of proteins in the range of 104–106, and duration of electrophoresis
Authors: Rothe Gunter M. , Purkhanbaba Huschang 
Published: May 2005

AbstractUntreated and processed gel plates of polyacrylamide (PAA) gradient gels were cut into strips perpendicularly to their length, and the wet and dry matter of the sections was determined. In untreated gels the apparent dry matter, as well as the relative dry matter, are a linear function of the gel length. In processed gels, however, only the apparent gel concentration increases linearly with the gel length, whereas the relative dry matter increases linearly with the square root of the gel length. The %T (content in polyacrylamide) was calculated from the apparent dry matter. The gel gradients used were found to be linear with respect to %T. Six different calibration proteins were run and their time‐dependent migration distances determined. For each protein a linear relationship exists between the square root of its migration distance (√D) and the logarithm of the electrophoretical duration (log t). In this relation √D can be substituted by the square root of the PAA concentration \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {\%{\rm T}} $\end{document} or by the relative dry matter (TM(%)). The resulting straight lines seem to intersect near the zero time of electrophoresis and the starting concentration of the gradient. At any time during electrophoresis (t» O), the square root of the migration distance (√D) of the calibration proteins and the logs of their Stokes' radii (log Rs), or the logs of their molecular weights (log MW), are correlated linearly. In this function, √D may be replaced by either \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {\%{\rm T}} $\end{document} or TM (%). It is shown that this correlation is not subject to time, although the constants are changed during the process of electrophoresis. The slopes of the lines and the duration of the electrophoresis (at a constant voltage) are related by a hyperbolic function. Regression lines obtained for different times during electrophoresis intersect in the range of the starting gel concentration. A more general equation is formulated which states ‐ within certain experimental limitations — the size of proteins, their migration distances, and the time of electrophoresis.
Preparative electrofocusing in agarose slab gels
Authors: Cantarow Walter D. , Saravis Calvin A. , Ives David V. , Zamcheck Norman 
Published: May 2005

AbstractA rapid preparative isoelectric focusing agarose slab gel technique that should prove to be valuable for the purification and characterization of clinically important macromolecules is described in this report. Up to 120 mg of plasma proteins have been focused in a 9.5 ml volume IsoGel agarose slab gel on a hydrophilic polyester plastic backing (GElBond) in 30 min or less. The agarose was permeable to proteins with molecular mass of 5–50 × 106 daltons and allowed recoveries of from 11–100 % depending on the proteins electrofocused. Proteins whose isoelectric points (pI) differed by 0.07 pH units will be separated by the described technique.
Nucleotide isotachophoretic assay: Method and application for determination of ATP/ADP ratio in several rat tissues
Authors: Pérez José A. , Mateo Fátima , Meléndez‐Hevia Enrique 
Published: May 2005

AbstractSeparation of ATP and ADP was achieved in isotachophoresis by using malonate, inorganic phosphate (Pi), creatine phosphate and lactate as spacers, which isolate these nucleotides from other UV‐absorbing products in rat tissues. By combining isotachophoresis with enzymatic end‐point analysis of several rat tissues, calibration for ATP and ADP was achieved obtaining an operative calibration curve that allows the use of isotachophoresis as the only analytical technique in further assays. ATP and ADP quantities of rat skeletal and smooth muscle, heart, liver, kidney and lung were determined by isotachophoresis. Individual variation was also calculated by analyzing 30 animals in the same control conditions, muscular tissues showing the greatest values for ATP and ADP, whereas kidney and lung show the smaller values. ATP/ADP ratio is discussed as a representative parameter for describing the energy content in biological tissues, there are also experimental reasons which imply that other product concentrations in this parameter not be included. This ATP/ADP ratio is calculated in the rat tissues studied, as well as its individual variation, obtaining the larger values for muscular tissues and the smaller for kidney. A great individual variation is found in kidney, followed by heart and skeletal muscle, and the most constant values are found in liver and smooth muscle. The results obtained in this calibration method for isotachophoresis assay suggest the use of a similar procedure in order to apply this technique for other nucleotide analysis.
Un‐supercoiled agarose with a degree of molecular sieving similar to that of crosslinked polyacrylamide
Authors: Buzás Zsuzsanna , Chrambach Andreas 
Published: May 2005

AbstractA previous observation by Nochumson et al. [7], showing that agarose after reduction or abolition of its supercoiled structure by hydroxyethylation (SeaPrep 15/45), exhibits a molecular sieving effect similar to polyacrylamide in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was confirmed and quantitated by Ferguson plot analysis in PAGE. SeaPrep 15/45 exhibits an effective fiber radius of 0.8 nm, as compared to 23.8 nm for native agarose and 0.4 nm for 2 % crosslinked polyacrylamide. Its effective pore size (estimated as \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {{\rm K}_{{\rm R polyacrylamide}} } /\sqrt {{\rm K}_{{\rm R agarose}} } $\end{document}) is 0.88 times that of polyacrylamide 2% crosslinked with N,N'‐methylene bisacrylamide (Bis). Although this is not a practical result as yet, due to the low melting temperature and poor gel strength of SeaPrep 15/45, it does sound the death knell for the use of crosslinked polyacrylamide in macromolecular separations, by demonstrating that a linear polymer capable of forming a gel on mere cooling is equivalent to the product of a laborious and relatively irreproducible free radical polymerization. It remains to find a linear polymer with higher melting point and better mechanical strength and adherence to glass walls than SeaPrep 15/45 to make crosslinked polyacrylamide obsolete.
Ultrathin‐layer isoelectric focusing of enzymes in liver samples of wagtails (Motacilla flava, ssp.)
Authors: Gemeiner Manfred , Miller Ingrid , Czikeli Harald 
Published: May 2005

AbstractWagtail samples (Motacilla flava, ssp; song bird) have been collected in different regios of central and southern Europe. Due to sampling extending over long periods of time it was necessary to investigate the effect of storing conditions (liquid nitrogen or ‐36 °C). Isozyme separation was done by polyacrylamide ultrathinlayer isoelectric focusing (100 μm). Liver homogenates were diluted in appropriate manner and 5 μl aliquots were used for separations. Gels for isoelectric focusing (T = 5.4, C = 2.8) containing different kinds of carrier ampholytec (Servalyt, Pharmalyte and Ampholines were cast on 12.5 × 20 cm pretreated glass plates according to the procedure of Radola [9]. Enzyme staining was performed in solution (ACP and esterases) or with an agarose overlay technique (LDH, ADH, MDH, PGM and GPI). Wagtail liver samples were chosen to optimize the electrophoretic conditions and to test the usefulness of different staining methods. This — and investigations presently under way on samples of pectoral muscle, heart, brain and blood — should allow us to elucidate genetic differences at the molecular level of the various subspecies of wagtails. The smaller total amount of protein available in some of these samples stresses the necessity of choosing a reproducible and sensitive, as well as rapid, electrophoretic technique.
Transferrin (Tf) subtypes in US Amerindians, Whites and Blacks using thin‐layer agarose gels: Report on a new variant TfC8
Authors: Dykes Dale D. , DeFurio Cathryn M. , Polesky Herbert F. 
Published: May 2005

AbstractUsing 0.5 mm agarose gels for Tf subtyping by isoelectric focusing we were able to identify ten variants at the Tf locus in US Amerindians, Whites and Blacks. The variant TfC4 was found only in the Amerindian populations. This population specific variant had gene frequncies ranging from 0.079 in the Walapi to 0.179 in the Apache. The Black sample studied had a lower incidence of TfC3 than in whites and was the only population with the marker TfD1. Thin agarose gels provided good separation of TfC3 from TfC1. Thin agarose gels provided good separation of TfC3 from TfC1 enabling us to demonstrate a new variant, TfC8, migrating between TfC1 and TfC3.
Diversification of isoelectric points from isolated chloroplasts during the cell cycle of Euglena gracilis, strain z
Authors: Brandt Peter , Ernst Dorothee 
Published: May 2005

AbstractWe describe a rapid method whereby isolated chloroplasts from Euglena gracilis can be separated in a Ficoll gradient according to their surface pK's by isoelectric focusing in one hour or less. The linear Ficoll gradient is made isotonic over the whole separation distance by addition of sucrose. No released proteins of the isolated chloroplasts are detectable after isoelectric focusing. Modifications of the chloroplast envelope during the cell cycle of Euglena gracilis are evident by alterations of their isoelectric points.
Isotachophoretic determination of a relative apparent mobility as an estimate of the electrophoretic mobility of ions
Authors: Carchon Hubert , Eggermont Ephrem 
Published: May 2005

AbstractAn isotachophoretic procedure for the estimation of the electrophoretic mobility of ions is described. The parameter, called apparent mobility (AM), is a function of the conditions of assay. Defining a relative apparent mobility (RAM) as the percentage value of the AM of the ion studied and the AM of a fully dissociated reference substance, an operational estimate of the electrophoretic mobility of ionic species is obtained. RAM values, therefore, are only a function of the leading ion and of the ion studied. If this ion demonstrates acid‐base equilibria, its RAM value will become pH dependent. As RAM values are independent on the conditions of assay, classification is meaningful. For a number of anions, RAM values may help to find out the optimal conditions for separation of ions by analytical isotachophoresis.
Preparative isotachophoresis in a flat bed of granulated gel. I. Principles and procedures, comparison with isoelectric focusing and application to the isolation of a low pI/high mobility form of cat liver cytosolic glutathione S‐transferase
Authors: Battersby Rüdiger V. , Holloway Christopher J. 
Published: May 2005

AbstractA method is reported for the separation and partial purification of proteins on a preparative scale (around 100 mg or more total protein per separation run), using the isotachophoretic principle. The technique was carried out using a granulated gel (Ultrodex) as supporting medium, with conventional flat‐bed electrophoresis equipment. This method was applied to the separation of multiple forms of glutathione S‐transferase, derived from the soluble cytosolic fraction of cat liver homogenate. Of particular interest in the present work was the isolation of the highest mobility form of the enzyme, which also functions as a cytosolic ligand‐binding protein for bilirubin. The advantages and disadvantages of isotachophoresis are compred with those of isoelectric focusing, using a simliar apparative set‐up. The latter had the primary drawback that some forms of the enzyme, particularly those with most acidic or alkaline isoelectric points, were, to a certain extent, irreversibly inactivated following focusing, whereas isotachophoresis yielded considerably higher volume activities and resulted in less inactivation during separation. The loss in activity of some forms of the enzyme was particularly marked in isoelectric focusing in thinner gels with agarose as supporting phase. Isotachophoresis has become associated with quite complicated instrumental requirements. The simple form of this technique reported in the present work should provide a useful addition to the spectrum of methods available for protein separation and purification. The major limitation of the flat‐bed configuration is that the actual resolution obtained cannot be fully exploited owing to the technical difficulties associated with sectioning the flat‐gel.
Enhancement of resolution in two‐dimensional gel electrophoresis and simultaneous resolution of acidic and basic proteins
Authors: Burghes Arthur H. M. , Dunn Michael J. , Dubowitz Victor 
Published: May 2005

AbstractIn studies of genetic diseases such as Duchenne muscular dystrophy (DMD), two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) provides an indispensable tool for studying protein changes. For optimum use of 2‐D PAGE it is important to maximize the resolution that can be obtained. In this study we have developed a system using flat‐bed isoelectric focusing (IEF) with gels bound to a plastic backing for the first‐dimension separation. This system, when compared to the conventional rod IEF system, was found to yield good resolution of both the acidic and basic proteins, which are normally separated using non‐equilibrium systems. We have also used the principals of gradient engineering in both the IEF and sodium dodecyl sulfate (SDS)‐PAGE dimensions to maximize the gel area used for the separations. Mixtures of various commercial carrier ampholytes were also used and were found to further enhance resolution.
Human leukemias: A preliminary 2‐D electrophoretic analysis
Authors: Lester Eric P. , Lemkin Peter F. , Lowery Joyce F. , Lipkin Lewis E. 
Published: May 2005

AbstractWe have investigated the qualitative and quantitative differences in the two‐dimensional (2‐D) gel fluorographic patterns of biosynthetically pulse‐labeled cellular proteins from the malignant cells of patients with various leukemias (acute myelogenous, acute lymphoblastic, chronic lymphocytic, and hairy cell). Significant similarities and differences between the major types of leukemias were found. An initial small data base was constructed using our GELLAB computer analysis system for 2‐D gels in order to define methods for data base definition, normalization, and visual verification of results. Search strategies to find significant spot differences, both qualitative and quantitative, between currently accepted classes of leukemias were explored. Potential methods of expressing in a summary statistic the overall similarities and differences between gels were also investigated. A series of proteins whose relative synthetic rates may serve as potential markers of states of differentiation in human leukemias were identified in this initial data base and are reported here.