bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrathin‐layer isoelectric focusing of enzymes in liver samples of wagtails (Motacilla flava, ssp.)
Authors: Gemeiner Manfred , Miller Ingrid , Czikeli Harald
Published: May 2005

Wagtail 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

Using 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

We 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

An 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

A 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

In 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

We 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.
Anomalous conductivity zones in electrophoresis. III. Experimental tests of the theory
Authors: Spencer Michael , Kirk Jean M.
Published: May 2005

An 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

We 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

The 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

The 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

The 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

Procedures 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

A 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

Three 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

A 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

Proteins 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

Alternative 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

The 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

Proteins 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

The 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

Silanization 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

As 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

A 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

Silver 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

A 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

Glycosaminoglycans, 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

A 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.
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

Recently, 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

Previously 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

A 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

The 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

The 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

Fluorescence 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

A 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

Two 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

The 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

Liver 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

The 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.
A modification for “smileless” polyacrylamide gels in nucleic acid sequencing
Authors: Brown Donald J. , Bos Timothy J.
Published: May 2005

The interpretation of nucleic acid sequences on polyacrylamide gels is often made difficult due to “smiles”. We have devised a simple modification for commonly used electrophoresis systems to eliminate this problem.
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

Lateral 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

We 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

The 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

An 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

A 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

The 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.