bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotyping of apolipoprotein E by immunoblotting in immobilized pH gradients
Authors: März Winfried , Cezanne Sabine , Groß Werner
Published: May 2005

An immunoblotting method for the determination of apolipoprotein E (apoE) phenotypes has been developed. Delipidated plasma proteins are focused in an immobilized pH gradient, and transferred to polyvinylidene difluoride (PVDF) membranes. ApoE isomorphs are identified by immunoperoxidase staining. The method allows reproducible assignment of apoE phenotypes without isolation of triglyceride‐rich lipoproteins. Only small amounts of serum are required. There are several important steps in the procedure: (i) delipidation is indispensable, (ii) carrier ampholytes have to be added to the gels and to the sample buffer, and, (iii) on immunostaining, polyvinylidene difluoride membrane provide an excellent signal‐to‐background ratio.
Resolving isoforms of aldose reductase by preparative isoelectric focusing in the Rotofor
Authors: Petrash J. Mark , Delucas Lawrence J. , Bowling Earnest , Egen Ned
Published: May 2005

We have resolved and characterized isoforms of aldose reductase from bovine and porcine lenses by preparative isoelectric focusing with narrow pH gradients using the Rotofor. Both bovine and porcine lens aldose reductases were resolved as two enzyme isoforms. The bovine isoforms were Mr40400 ± 445 polypeptides of pI 4.71 and 5.19. Porcine isoforms were Mr 41500 ± 450 polypeptides of pI 4.90 and 5.30 Staphylococcus aureus V‐8 protease digestion patterns for each set of isoforms were essentially identical and all isoforms probably contain blocked amino terminal amino acids. Antiserum to bovine lens aldose reductase cross‐reacted with porcine lens aldose reductase. Each isoform displayed substrate preferences characteristic of mammalian aldose reductases. With purification, both bovine and porcine lens aldose reductases became less senstive to inhibition by 6‐fluoro‐spiro‐(chroman, 4,4′‐imidazolidine)‐2′,5′‐dione (sorbinil).
The grammatical rule for all DNA: Junk and coding sequences
Authors: Ohno Susumu , Yomo Tetsuya
Published: May 2005

Selfish DNA, coding sequences, and junk DNA in the genome are no stranger to each other; rather, they represent three phases in the life cycle of DNA. Accordingly, they all obey the same grammatical rule of TG/CA/CT excess and CG/TA deficiency. On the one hand, it is this very rule which keeps isoelectric points of most proteins near the neutral range. On the other hand, this rule creates numerous palindromes, thus maintaining symmetry between complementary strands. Many of these palindromes encode identical oligopeptides on both strands.
Isolation of simple‐sequence loci for use in polymerase chain reaction‐based DNA fingerprinting
Authors: Rassmann Kornelia , Schlötterer Christian , Tautz Diethard
Published: May 2005

Simple sequences are short regions of tandem repetitions of mono‐, di‐, tri‐, or tetranucleotide motifs and occur as repetitive elements in all eukaryotic genomes. These regions tend to be hypervariable in length and can therefore be exploited for DNA fingerprinting purposes, using the polymerase chain reaction with primers flanking such regions. We describe how suitable simple‐sequence loci can be isolated from any given eukaryotic DNA. We show the DNA sequences for a number of variants of such loci and discuss the current results on their usefulness for DNA fingerprinting.
Detection of single and multiple polymorphic loci by synthetic tandem repeats of short oligonucleotides
Authors: Vergnaud Gilles , Mariat Denis , Zoroastro Monique , Lauthier Valérie
Published: May 2005

Loci containing tandem repeats of short sequences are sometimes associated with a high level of polymorphism due to variations in the number of repeats. The different variants can be easily characterized by Southern blotting when the repeats span a range from a few hundred bases to a few kilobases, and probes derived from such tandem repeats constitute convenient genetic markers. These structures, usually called minisatellites, are best documented in the human genome, where their number has been estimated to be at least 1500. However, their role and mode of evolution are poorly understood. We are developing tools to evaluate the number of such redunddant sequences in a genome and to gain access to new polymorphic loci. Our strategy is based on the use of polymers of oligonucleotides as DNA probes for hybridization on Southern blots. In a previous report, we made polymers with random units of 14 bp and showed that they detect multiple polymorphic loci on human genomic DNA. At present, we are testing the effect of an increase in the complexity of the polymer, as obtained by the use of a longer random unit, and the effect of slight sequence modifications to a particular tandem repeat sequence. In addition, some of these synthetic probes can detect a single polymorphic locus and directly provide new genetic markers.
DNA bioprints: Simple nonisotopic DNA fingerprints with biotinylated probes
Authors: Pena Sérgio D. J. , Macedo Andrea M. , Gontijo Nelder F. , Medeiros Arnaldo M. , Ribeiro Julio Cesar C.
Published: May 2005

The discovery of multilocal DNA fingerprinting represented a revolution in criminal identification and paternity testing. However, for routine use in clinical laboratories, the standard DNA fingerprint methodology is too complex. We have been successful in the development of a simplified DNA nonisotopic fingerprinting system using biotin‐labeled probes which we have called DNA bioprints. To achieve this we explored three main technical features: utilization of biotinylated nonradioactive probes as a simpler substitute for 32P‐labeled probes, utilization of oligonucleotide probes as a simpler substitute for recombinant probes, and direct hybridization in the dried agarose gel as a simpler substitute for Southern blots. In this article we review our results in the development of DNA bioprints.
DNA fingerprinting in domestic animals
Authors: Buitkamp Johannes , Ammer Hubert , Geldermann Hermann
Published: May 2005

DNAs of several species of domestic animals digested with the restriction endonucleases HinfI, AluI and HaeIII were hybridized with different synthetic probes. DNA fingerprint patterns were found in each investigated species by at least two of these probes. Furthermore, two probes gave sex‐specific banding patterns in the chicken. Some applications of DNA fingerprinting in domestic animals are discussed.
Forensics of birds of prey by DNA fingerprinting with 32P‐labeled oligonucleotide probes
Authors: Wolfes Rüdiger , Máthé Judith , Seitz Alfred
Published: May 2005

Paternity tests on confiscated families of eight species of birds of prey were carried out successfully by DNA fingerprinting with 32P‐labele oligonucleotide probes. Variations in the number of hybridized fragments, depend ing on the species of birds, are observed using the same probe, as well as differences of polymorphism by hybridizing the DNA samples with several oligonucleotide probes.
Monitoring genomic alterations with a panel of oligonucleotide probes specific for various simple repeat motifs
Authors: Nürnberg Peter , Barth Ingrid , Fuhrmann Eike , Lenzner Claudia , Losanova Tanka , Peters Christian , Pöche Hubert , Thiel Gundula
Published: May 2005

Germline and somatic instability of the human genome was studied, using synthetic oligonucletides specific for simple repeat motifs. The following probes were used: (GTG)5, (GACA)4, (GATA)4, (CT)8, (TTAGGG)3, (GT)8, (GAA)6 and (GGAT)4. Each of them is unique with respect to the target regions recognized in the genome. Thus compilation of the various fingerprint data provides a complex map of the genome (and its deviations). While the fingerprints of differentiated somatic tissues never showed any alterations, in tumor tissues (namely gliomas) many changes could be detected. Most of the latter reflect secondary karyological aberrations. In nearly one third of the gliomas, drastically amplified and apparently monomorphic DNA fragments were identified. This marker should make it possible to deal with causal pathogenetic mechanisms as well as novel diagnostic strategies.
Sizing bands on autoradiograms: A study of precision for scoring DNA fingerprints
Authors: Galbraith David A. , Boag Peter T. , Gibbs H. Lisle , White Brad N.
Published: May 2005

We replicated DNA fingerprints of snapping turtles (Chelydra serpentina) and hypervariable restriction fragments of red‐winged black birds (Agelaius phoeniceus) to estimate the between‐blot and between‐lane components of variance in molecular weights of restriction fragments. Molecular weight standards were included in every lane, and bands were sized using a sonic digitizer. In both studies, a strong positive correlation was found between band size and coefficient of variation (CV; mean = 0.7 %). In the DNA fingerprint study, 26 % of the variance in estimates of band size was due to differences between blots, 10 % due to differences between lanes on the same blot, and 64 % due to error in the digitizing process. In the restriction fragment length polymorphism (RFLP) study, 16 % of the variance was due to difference between lanes, and 84 % to digitizing. Statistical models were developed to measure the effect of sizing error on identifying identical fragments in different lanes or on different blots, in categorizing distinct alleles, and in determining the size of bins in operational allele definitions. We suggest that the distance between bands be at least 2.8 standard deviations (SD) before they are declared different at α = 0.05, and 3.7 SD for α = 0.01. A variation in CVs strongly indicate that empirical relationships between SD and band size must be used to decide if two bands represent the same allele. Alleles must be at least 3.9 SD apart before the chance of assigning new observations in error falls below 0.05. We suggest that a minimum bin width of 16 SD is necessary before the chances of assigning a band to the wrong bin falls below 0.05.
Resolution of glycogen phosphorylase isoenzymes in precast Phastsystem polyacrylamide gels
Authors: Mayer Doris , Letsch Iris
Published: May 2005

Homogeneous (7.5 %) and gradient (10–15 %) ultrathin nondenaturating miniaturized polyacrylamide gels (Pharmacia PhastGel media) were used to separate glycogen phosphorylase isoforms from rabbit muscle, rat liver and brain, MH 3924A cells, a dedifferentiated hepatocellular carcinoma of the rat, and C1I cells, a nontumorigenic epithelial rat liver cell line. The enzymes were detected by in situ phosphorylase assay and by immunoblotting. Phosphorylase proteins from the brain, MH 3924A, and C1I exhibited similar electrophoretic mobility, which was different from that of the enzymes from the muscle and normal liver. Molecular weight determination from sodium dodecyl sulfate gels yielded similar data for the subunits of muscle and liver enzymes (98 000 and 96 000), respectively, on one hand, and brain, MH 3924A tumor, and nontumorigenic C1I cells (93 000, 93 000 and 92 000), respectively, on the other. In the native gels a enzymes migrated as dimers: for muscle phosphorylase a, a tetramer was also observed. The a and b forms of the enzymes could not be resolved. An antibody raise against rat liver phosphorylase reacted only with the liver enzyme, whereas an antibody raised against brain phosphorylase stained the brain enzyme and the enzymes from MH 3924A and C1I cells. This indicates that hepatoma cells and immortalized nontumorigenic epithelial liver cells express a phosphorylase isoenzyme that is different from the liver type but similar to the brain type. The PhastSystem provides a rapid, sensitive, and highly reproducible method to resolve the different isoenzymes of glycogen phosphorylase.
High resolution electrophoresis of soybean seed isoesterases
Authors: Wiesner Ivo
Published: May 2005

A modified discontinuous buffer system for vertical polyacrylamide electrophoresis was developed with large changes in component concerntration from the original Tris‐HCI/Tris‐glycine system of the Ornstein‐Davis type. The buffer system is able to resolve the fast soybean seed isoesterases crucial for cultivar discrimination. A higher number of anodic bands is obtainedin comparison with separations under standard conditions. A “gelating boundary”, resulting from the aggregation of seed proteins, is retarded and does not migrate out of the upper stacking gel. The same buffer modification is also beneficial to the separation of native seed proteins.
Two‐dimensional electrophoresis of human parotid salivary proteins from normal and connective tissue disorder subjects using immobilised pH gradients
Authors: Beeley Josie A. , Khoo Kong Soo , Lamey Philip‐John
Published: May 2005

Two‐dimensional electrophoretic analysis of human salivary proteins using immobilised pH gradients in the first dimension, thin‐layer gradient horizontal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the second, and modified staining procedures has resulted in a substantial improvement in their resolution. Unlike carrier ampholyte‐based techniques, immobilised pH gradients prevent the loss of proteins of pI>8; accordingly, basic components, including basic proline‐rich proteins, can now be resolved. A two‐dimensional map showing the locations and identities of most of the major proteins has been constructed. Narrow‐range pH gradients can be constructed to give increased resolution of proteins of particular interest. By means of a pH 3.5–5.0 gradient, the abnormal salivary proteins associated with connective tissue disorders were found to be a highly heterogeneous group of pI ∼ 3.75–4.75 and Mr ∼ 32 000; although low levels occurred in some normal individuals, there was less heterogeneity (pI∼3.75–4.25). The technique should form a base for future structural, functional, and clinical studies on human salivary proteins.
Nuclear and DNA‐binding proteins in human brain tumors
Authors: Müller Andreas , Henn Wolfram , Unteregger Gerhard
Published: May 2005

Nuclear proteins obtained from human brain tumor cell lines by differential salt extraction were subjected to high‐resolution two‐dimensional electrophoresis. Several hundred spots were detectable in the low salt (0.4 M NaCl) extract using silver staining. These patterns exhibited remarkable differences between the different cell lines we analyzed. A less complex pattern occured when nuclei were subsequently treated with high salt (2.5 M NaCl/5 M urea). We compared the electropherograms from various human glioblastoma cell lines and found them very, similar and even a high degree of similarity occurs between glioblastomas and other human tumor cell lines. Beside these more general observations we detected several proteins at least enriched in human glioblastomas which were, totally absent in low grade astrocytomas and nonglial tumors. They could be separated from the bulk of nonspecific proteins by simple modifications of the isoelectric focusing conditions. From these results we conclude that nuclear proteins obtained by sequential salt extraction and separated by two‐dimensional techniques may provide tumor specific proteins suitable for antibody production.
Molecular profiles of the cell membrane bound and cytoplasmic forms of the human MHC Class‐II associated invariant polypeptides
Authors: Fernandez Nelson , Klidis Aristea
Published: May 2005

Several sub‐types of histocompatibility class‐II molecules are known to exist, including DR, DQ and DP, each subset organized in alpha and beta heterodimers. These molecules play a central role in immunorecognition via direct noncovalent binding of degraded nonself immunogenic peptides followed by ligand‐interaction with T cells. In the cytoplasm the class‐II alpha and beta chains associate, in addition with a third backbone molecule known as the invariant chain. In this study we show that all class‐II subsets associate with the invariant chain probably from the same pool and that a subset of invariant chain is expressed as a cell surface subunit. This sub‐unit, whose function is still unknown, was detected by flowcytometry and by two‐dimensional gel analysis followed by antibody‐mediated immunoprecipitations.
Biochemical characterization by two‐dimensional electrophoresis of lymphocyte antigens involved in cell‐to‐cell or cell‐to‐matrix adhesion
Authors: Zocchi Maria Raffaella , Fabbri Monica , Poggi Alessandro , Gianazza Elisabetta
Published: May 2005

We have exemplified three cases of application of two‐dimensional (2‐D) electrophoresis to the characterization of lymphocyte membrane antigens. We could show that the proteins recognized by two monoclonal antibodies, LAK1 and LAK2, on the surface of large granular cells mediating natural‐ and lymphokineactivated killing are distinct molecules. LAK1 is expressed without any structural modification, even on the surface of endothelial cells. Another membrane antigen, recognized by the monoclonal antibody FB 12, was shown to have the overall structure of the integrins of the very late activation (VLA) class, being composed of an α and of a β subunit. The latter corresponded to the β1 type as already characterized for other VLAs, whereas the α chain was different from α1 through α6. The 2‐D protocol using immobilized pH gradients for the first dimension allows reliable assessment of the identity of individual components because of the reproducibility of the absolute coordinates for spot position.
Production of high purity proteins by elution of individual fractions from two‐dimensional gels
Authors: Tsvetkova Marina N. , Kovalyov Leonid I. , Laptev Alexei V. , Shishkin Sergei S.
Published: May 2005

Results are presented on the production of purified myocardial proteins from two‐dimensional (2‐D) gels. Proteins were fixed in a native condition using potassium acetate and then eluted into an aqueous solution. Homogeneous tropomyosin and myosin light chain fractions and a number of nonidentified myocardial proteins present on 2‐D gels were obtained.
Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide
Authors: Holmes Diana L. , Stellwagen Nancy C.
Published: May 2005

The mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N′‐methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5–10.5%T (w/v); the Bis concentration ranged from 0.5–10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5–3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at ∼ 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77–90). In gels containing 3–10%C, the calculated pore radii decreased with increasing gel concentration approximately as T−0.5, suggesting that these gels may be long fiber or “1‐D” gels. The pore radii of gels with lower crosslinker concentrations decreased more slowly with increasing polyacrylamide concentration. Pore sizes were also determined for gels containing 3% Bis and 0.5% w/v agarose, and 3% Bis plus 2% w/w high molecular weight linear polyacrylamide. The pore radii of the mixed agarose‐polyacrylamide gels were only about half as large as those observed in gels of the same composition without agarose. The inclusion of 2% w/w linear polyacrylamide in the polymerization mixture decreased the effective pore size of the polyacrylamide matrix by about 10%.
Isoelectric membrane simulator: A computational approach for isoelectric Immobiline membranes
Authors: Tonani Carlo , Faupel Michel , Righetti Pier Giorgio
Published: May 2005

Isoelectric membrane simulator (IMS) is a computer program meant for computation of pH, buffering power (β), ionic strength (I) and dissociation degree (α) of a mixture of up to 3 buffering and 1 titrant Immobilines, for generating in a reproducible manner isoelectric membranes. Such membranes, of precise isoelectric point, are then used for large‐scale protein purification by isoelectric focusing in multicompartment electrolyzers. IMS can be used, in a more general application, for titrating mixtures of buffers to a desired pH value. This versatile program is written in M. Q. BASIC rel.2.5 and it runs on any IBM hardware or compatible machine supported by MS‐DOS. An example of purification of superoxide dismutase in a multicompartment electrolyzer with a set of fixed pI membranes of widely differing composition is shown.
A modified method of genomic DNA preparation in agarose inserts for pulse field gel electrophoresis
Authors: Poddar Saibal K.
Published: May 2005

According to standard protocol, DNA in agarose inserts is prepared by first embedding the Cell in agarose. This is then incubated in the required enzyme (lysozyme, lysostaphin, or zymolase) depending on the Cell type (bacterial or plants), for spheroplast formation. Subsequent treatment of the spheroplast with protein ase K allows the isolation of large genomic DNA in agarose suitable for pulse field gel Electrophoresis. An efficient and rapid method of preparation of spheroplast is described. In this method a low concentration of enzyme required for spheroplast formation was added before embedding the Cell in agarose, which facilitated the digestion of Cell wall by the enzyme and allowed use of a low amount of enzyme. Digestion of DNA in agarose inserts prepared by this method, with rare cutting restriction enzyme and pulse field gel Electrophoresis, showed that the quality of DNA was as good as obtained by the standard method.
Detection of proteins after immunoblotting on nitrocellulose using fluorescent antibodies
Authors: Abuharfeil Nizar M. , Atmeh Ragheb F. , Abo‐Shehada Mahmoud N. , El‐Sukhon Saeb N.
Published: May 2005

A Western blot method is described utilizing 5‐dimethylaminonaphthalene‐1‐sulfonyl (dansyl chloride)‐conjugated antibodies to detect proteins transferred onto nitrocellulose. Four proteins of different molecular weight, including human albumin, gamma heavy chain, kappa and lambda light chains of immunoglobulins, were tested. Labeling with the fluorochrome was compared with the method of enzyme‐conjugated antibodies and was found to have similar sensitivity, specificity, and safety. Moreover, this method is less expensive, and the labeled antibodies can be prepared fresh in a short time and are free from the disadvantages of enzyme label whose activity is affected by the presence of activators or inhibitors in the tested specimens.
Procedures and computer program for deriving the Ferguson plot from electrophoresis in a single pore gradient gel: Application to agarose gel and a polystyrene particle
Authors: Tietz Dietmar , Gombocz Erich , Chrambach Andreas
Published: May 2005

This study presents a computerized evaluation of pore gradient gel electrophoretograms to arrive at estimates for both the particle‐free mobility and retardation coefficient, which is related to particle size. Agarose pore gradient gels ranging from 0.2 to 1.1% agarose were formed. Gel gradients were stabilized during their formation by a density gradient of 0–20% 5‐(N‐2, 3‐dihydroxypropylacetamido)2, 4, 6‐triiodo‐N,N′ bis‐(2, 3‐dihydroxypropyl)‐isophthalamide (Nycodenz). Densitometry of gelled‐in Bromophenol Blue showed that these pore gradients exhibited a linear central segment and were reproducible. Migration distances of polystyrene sulfate microspheres (36.5 nm radius) in agarose pore gradient gel electrophoresis were determined by time‐lapse photography at several durations of electrophoresis. These migration distances were evaluated as a function of migration time as previously reported (D. Tietz, Adv. Electrophoresis 1988, 2, 109–169). Although this is not necessarily required, the mathematical approach used in this study assumed linearity of both the pore gradient and the Ferguson plot for reasons of simplicity. The data evaluation on the basis of the extended Ogston model is incorporated in a user‐friendly program, GRADFIT, which is designed for personal computers (Macintosh). The results obtained are compared with (1) conventional electrophoresis using several gels of single concentration with and without Nycodenz, and (ii) a different mathematical approach for the analysis of gradient gels (Rodbard et al., Anal. Biochem. 1971, 40, 135–157). Moreover, a simple procedure for evaluating linear pore gradient gels using linear regression analysis is presented. It is concluded that the values of particle‐free mobility and retardation coefficient derived from pore gradient gel electrophoresis using the different mathematical methods are statistically indistinguishable from each other. However, these values are different, albeit close, to those obtained from conventional Ferguson plots. One of the possible reasons for this relatively minor discrepancy is that the particle‐free mobility changed slightly during electrophoresis, which has a different effect on electrophoresis in homogeneous gels (single time measurement) and pore gradient gels (multiple time measurements). The characterization of particles according to size and charge by pore gradient electrophoresis provides a significant operational simplification and sample economy compared to that requiring the, use of several gel concentrations, although at the price of increased requirements of instrumentation.
Implementation of a two‐dimensional electrophoresis related laboratory information processing system: Database aspects
Authors: Ali Imtiaz , Chan Yishin , Kuick Rork , Teichroew Daniel , Hanash Samir M.
Published: May 2005

A laboratory information processing system (LIPS) has been developed to provide support for various laboratory activities related to two‐dimensional electrophoresis. In this paper we present the relational schema for each level of activity and the application development of LIPS, in terms of the relational database management system being used. We also discuss our experience with the current system.
A comprehensive two‐dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: Towards an integrated approach to the study of cell proliferation, differentiation and skin diseases
Authors: Celis Julio E. , Madsen Peder , Rasmussen Hanne H. , Leffers Henrik , Honoré Bent , Gesser Borbala , Dejgaard Kurt , Olsen Eydfinnur , Magnusson Nils , Kiil Jette , Celis Ariana , Lauridsen Jette B. , Basse Bodil , Ratz Gitte P. , Andersen Annette H. , Walbum Else , Brandstrup Birgitte , Pedersen Peter S. , Brandt Niels J. , Puype Magda , Van Damme Josef , Vandekerckhove Joël
Published: May 2005

A two‐dimensional (2‐D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine‐labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer‐aided 2‐D gel electrophoresis. The protein numbers in this database differ from those reported in an earlier version due to changes in the scanning hardware (Celis et al., Electrophoresis 1990, 11, 242–254). Annotation categories reported include: “protein name” (listing 207 known proteins in alphabetical order), “basal cell markers”, “differentiation markers”, “proteins highly up‐regulated in psoriatic skin”, “microsequenced proteins” and “human autoantigens”. For reference, we have also included 2‐D gel (isoelectric focusing) patterns of cultured normal and psoriatic keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2‐D gel protein database will be updated yearly in the November issue of Electrophoresis.
Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of human parotid salivary proteins
Authors: Beeley Josie A. , Sweeney Dan , Lindsay Joan C. B. , Buchanan M. Luise , Sarna Lalita , Khoo Kong Soo
Published: May 2005

The proteins in human parotid saliva have been separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis into 20 or more well resolved species. The Coomassie Brilliant Blue (CBB) R‐250 and silver staining procedures have been modified to overcome the problems encountered with staining of praline‐rich proteins. By means of the CBB R‐250 procedure which stains praline‐rich proteins pink‐violet, immunoblotting, concanavalin A binding, periodate‐Schiff staining and zinc binding, all of the major proteins have been characterised. Substantial individual‐to‐individual differences were observed in the protein patterns formed. Comparison of parotid, submandibular, and whole saliva from a single individual indicated that fewer proline‐rich proteins are expressed in submandibular saliva than in parotid, but whole saliva contains much lower levels than either duct secretion. The results will form a useful base for future research into the functions of salivary proteins.
Determination of rat plasma esterase‐1 (ES‐1) activity by scanning densitometry of gradient polyacrylamide gels with zymogram detection
Authors: Van Lith Hein A. , Haller Manuela , Van Zutphen Bert F. M. , Beynen Anton C.
Published: May 2005

There is no specific assay for rat plasma esterase‐1 (ES‐1) activity. Plasma contains many esterases, while known substrates do not discriminate between esterases. With gel electrophoresis, plasma esterase isozymes can be separated. Thus, a method consisting of gradient polyacrylamide gel electrophoresis, visualization of the enzyme with a staining technique based on substrate conversion, and densitometric scanning of the stained gel has been developed for quantitative measurement of rat plasma ES‐1 activity. ES‐1 activities were expressed as total peak areas. Reproducibility of the method was found to be about 10% (expressed as apparent between‐gel coefficient of variation). When the ES‐1 zone areas was expressed relative to that of a plasma ES‐1 standard, reproducibility was about 3%. The kinetics of catalysis of α‐naphthyl acetate hydrolysis by ES‐1 could be determined with the gel scanning assay; the Km was 0.76 mm. At the α‐naphthyl acetate concentration of 2.69 mm, total peak areas of the ES‐1 zone were linearly associated with the staining time (up to at least 40 min) and amount of plasma (up to 26.25 μL). The pH of the staining buffer influences the ES‐1 zone area, the largest areas being obtained when the pH ranged between 7.0 and 7.8. With propionate as acyl moiety of the α‐naphthyl ester substrate, ES‐1 zone areas were higher than with either acetate, butyrate or hexanoate.
A new staining method for the detection of activities of H2O2‐producing oxidases on gels and blots using cerium and 3,3′‐diaminobenzidine
Authors: Seitz Jürgen , Keppler Claudia , Fahimi H. Dariush , Völkl Alfred
Published: May 2005

Cerium chloride (CeCl3) was used to trap the hydrogen peroxide generated by several oxidases on native gels and blots. The pale yellow color of cerium perhydroxide formed is converted to a brown‐black precipitate by the subsequent reaction with 3.3′‐diaminobenzidine. The suitability of this method for the detection of the activity of several oxidases on gels and on blots under nondenaturing conditions, employing different electrophoretic systems and resolving techniques, is demonstrated. Moreover, this method has proven to be highly suitable for the assessment of the substrate and stereospecifity of oxidases, the determination of their molecular weights, and the isoelectric points of isoforms.
Pressure refilled polyacrylamide columns for the separation of oligonucleotides by capillary electrophoresis
Authors: Sudor Jan , Foret František , Boček Petr
Published: May 2005

The separation of oligonucleotides by capillary zone electrophoresis (CZE) was studied in fused silica separation capillaries filled by linear (noncrosslinked) polyacrylamide (PAA) solutions, introduced into the capillary from the stock by pressure after each analysis. The time‐consuming in‐capillary polymerization step could thus be avoided, and fast and reproducible repetition of the analyses was assured. The PAA concentrations varied within the range of 3–10% and both the reproducibility of the analyses and the stability of the solution in the capillary, with and without a chemically treated inner wall, were tested. Ferguson plots were used to assess the size selectivity of the separation.
High speed electrophoresis simulation for optimization of continuous flow electrophoresis and high performance capillary techniques: Part I. Computer model
Authors: Heinrich Jörn , Wagner Horst
Published: May 2005

A computer program for high‐speed simulation and optimization of electrophoretic processes has been developed for carrier‐free systems of all kinds. The calculations are based on the one‐dimensional dynamic (transient‐state) model. The three‐dimensional geometry of the simulation space can be chosen deliberately. With a highly efficient transport algorithm instead of complicated integration schemes for the transport equations, the calculation time can be effectively spent on various important parameters such as ionic strength, temperature, Joule heat, activity coefficients and concentration changes due to membranes. The parameter set of any carrier free electrophoretic method (i.e., continuous‐flow electrophoresis, capillary isotachophoresis and high performance capillary zone electrophoresis) can be imported directly into the computer program by means of a graphic user interface. The program performs overnight‐simulation of any electrophoretic system containing up to 15 components.
Formamide modified polyacrylamide gels for DNA sequencing by capillary gel electrophoresis
Authors: Rocheleau Marie J. , Grey Ronda J. , Chen Da Yong , Harke Heather R. , Dovichi Norman J.
Published: May 2005

Compressions are occasionally found during the separation of DNA sequencing fragments, particularly in G/C‐rich regions and in gels operated at room temperature. Addition of at least 10% formamide to urea/polyacrylamide sequencing gels improves the denaturing capacity of the gel, minimizing compressions. Addition of 20% or more formamide decreases the separation rate, theoretical plate count, and resolution for normally migrating fragments. An optimum concentration of 10% formamide improves resolution of compressed regions without degrading the other characteristics of the gel. Operation of gels at room temperature simplifies the engineering associated with automated sequencers based on capillary gel electrophoresis.
Pulsed field sequencing gel electrophoresis
Authors: Brassard Eric , Turmel Chantal , Noolandi Jaan
Published: May 2005

The effect of pulsed fields on sequencing gel electrophoresis is investigated, using DNA fragment markers ranging in size from 20 to 6557 bases. For high continuous electric fields (5000 V/55 cm) band inversion is observed in which fragments larger than 4000 bases migrate faster than those of 800–1000 bases. The use of one‐dimensional pulsed field gel electrophoresis (ODPFGE) eliminates band inversion and extends the monotonic size‐mobility relationship of the DNA markers up to about 4000 bases. The relevance of these results, obtained using a manual sequencing process with autoradiographic detection, to automated sequencers is discussed.
Analysis of members of the Ig‐gene superfamily by thermal gradient polyacrylamide gel electrophoresis
Authors: Tulp Abraham , Verwoerd Desireé
Published: May 2005

A solid aluminum block, connected with a warm and cold thermostated waterbath, provided for a linear transversal temperature gradient (TG) during polyacrylamide gel electrophoresis (PAGE). Noncovalently bound heavy chain dimers as well as heavy‐light chain dimers, derived from human monoclonal IgG, could be melted into monomers using a 40–75°C TG under conditions of sodium dodecylsulfate‐PAGE. Using native PAGE, majorhistocompatibility complex (MHC) class I molecules, preloaded with the iodinated peptide FAPGNYPAL could be melted in a 4–40°C TG to release the peptide. The method is in general applicable to thermal stability analysis of noncovalently bound hetero‐oligomers if the product after melting possess different electrophoretic mobilities.
Thin‐layer electrophoresis with PhastSystem facilitates analysis of phosphoamino acids from proteins bounc to Immobilon
Authors: Lippmann Corinna , Lindschau Carsten , Erdmann Volker A.
Published: May 2005

A modification of thin‐layer electrophoresis of phosphoamino acids on miniplates, using the PhastSystem (Pharmacia) is described. This method not only reduces the amount of radioactivity required but also the radioactive waste. In addition, it also decreases the handling of hazardous substances and takes only a fraction of the time required for the standard procedure.
Preparative continuous separation of biological particles by means of free‐flow magnetophoresis in a free‐flow electrophoresis chamber
Authors: Hartig Roland , Hausmann Michael , Schmitt Joachim , Herrmann Dieter B. J. , Riedmiller Michael , Cremer Christoph
Published: May 2005

For sorting, cells or cellular components can specifically be labeled by antibody‐coated magnetic beads. We have developed a device for continuous magnetic sorting based on the flow‐chamber of a free‐flow electrophoresis system. Magnetically labeled particles are injected into a given continuously flowing chamber buffer and pass an inhomogeneous magnetic field, configurated perpendicular to the flow direction. According to its magnetic moment, the magnetic material is deviated into the direction of the magnetic forces, while nonmagnetic material passes the field without interaction. The magnetic forces can be changed with the electrical current of the solenoids producing the magnetic field. As in the free‐flow electrophoresis system, the particle fractions are collected in different vials. Online control of the experiments can be performed by an optical scanning system. Experiments with model particles achieved a sorting purity of more than 99% at a rate of up to 5 × 108particles per hour. In experiments with blood cells, a high enrichment of either B‐or T‐lymphocytes was obtained. In contrast to free‐flow electrophoresis, there is no limitation, in principle, regarding the type of chamber buffer to be used. This allows an optimal adaptation of the buffer conditions to the requirements of vital sorting. The preliminary results so far confirm this conclusion.
Application of capillary electrophoresis in peptide research
Authors: Yildiz Eraslan , Grübler Gerald , Hörger Susanne , Zimmermann Hans , Echner Hartmut , Stoeva Stanka , Voelter Wolfgang
Published: May 2005

Comparison of high performance liquid chromatograms (HPLC) with capillary electrophoresis shows the latter to be superior in many cases owing to rapid separation, high resolution and high sensitivity. This is demonstrated with two examples (i) the isolation of natural peptides from bovine tissue and (ii) characterization of a synthetic peptide mixture with the natural sequence (fragment) of the human immunodeficiency virus (HIV) transmembrane glycoprotein gp 41 env.
Improving the detection of proteins after transfer to polyvinylidene difluoride membranes
Authors: Sanchez Jean‐Charles , Ravier Florence , Pasquali Christian , Frutiger Séverine , Paquet Nicole , Bjellqvist Bengt , Hochstrasser Denis F. , Hughes Graham J.
Published: May 2005

N‐Terminal sequence analysis of proteins separated by two‐dimensional polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes has become the method for molecular characterization of proteins contained in biological samples. However, the proteins of lower abundance cannot be sequenced directly, without improving the technique. We have studied a drying method on several PVDF membranes including Trans‐Blott, Immobilon P and Problott. Using Amido Black, Coomassie Brilliant Blue R‐250 and Ponceau S, we have obtained, in comparison with the non‐dried membranes, an enormous increase in the number of detectable proteins.
Examination of microheterogeneity in grass pollen allergens
Authors: Petersen Arnd , Becker Wolf‐Meinhard , Schlaak Max
Published: May 2005

The separation of timothy pollen extract by two‐dimensional immunoblotting revealed microheterogeneity of the major allergens PhI p I and PhI p V. There was not only a diversity in size, 38 and 32 kDa for PhI p V and 37,35, and 32 kDa for PhI p I, but also a separation into proteins of identical sizes but different pIs. Since former studies on the protein structure by amino acid analysis and N‐terminal micro‐sequencing did not reveal any differences, we examined other possibilities that might cause microheterogeneity. In allergens belonging to the PhI p I group, the variability in pI can be due to the carbohydrate structure and to the fact that charges are hidden in the interior of the protein, as shown by varying concentrations of urea. On the other hand we were not able to detect any such reasons for the existence of PhI p V isoallergens. Thus, we assume that they are stable conformational isomers of the proteins (allomorphism) or that there are only slight variations in the internal sequences of these proteins (polymorphism) causing distinct pIs.
Detection of polypeptides and amylase isoenzyme modifications related to malting quality during malting process of barley by two‐dimensional electrophoresis and isoelectric focusing with immobilized pH gradients
Authors: Görg Angelika , Postel Wilhelm , Weiss Walter
Published: May 2005

Two cultivars (“Alexis” and “Lenka”) of contrasting final attenuation values were malted, and the protein and amylase isoenzyme composition, as well as the change in protein and amylase isoenzyme composition during malting, was investigated by two‐dimensional polyacrylamide gel electrophoresis of total proteins, and isoelectric focusing of amylase isoenzymes, respectively. Isoelectric focusing demonstrated that significant differences exist between the amylase isoenzyme patterns of the two cultivars, suggesting a correlation between the presence of certain amylase isoenzyme bands and final attenuation. This finding was confirmed by analysis of 36 barley cultivars with a wide range of quality. It was shown that all cultivars which are of low or, at best, moderate final attenuation values exhibit the amylase band “B” (isoelectric point ≈︁ 6.8), whereas those cultivars which are predominantly of high malting grade do not possess this “B” isoenzyme band, but exhibit the pronounced “A” isoenzyme band (isoelectric point ≈︁ 6.5) instead, suggesting that these isoenzymes (which we suppose to be β‐amylases) can be utilized to predict the final attenuation values of unknown barley samples or new lines. However, “final attenuation” is a complex function. Preliminary results of two‐dimensional gel electrophoresis indicate that other factors, such as total amount of amylases, or a 19 kDa A hordein‐like polypeptide, which was degraded faster in the low malting grade cultivar “Lenka”, may also have a role in determining quality.
Improved phenotyping of alpha1‐antichymotrypsin (ACT) by isoelectric focusing and immunoprinting: First demonstration of a deficient protein variant in the ACT system
Authors: Weidinger Sebastian , Poller Wolfgang , Faber Jakob‐Peter , Schwarzfischer Friedrich
Published: May 2005

Genetic variation of human alpha1 ‐antichymotrypsin (ACT) was investigated in sera using thin‐layer polyacrylamide gel isoelectric focusing (pH range 4.0–6.5) followed by immunoprinting with a monospecific anti‐human ACT antibody. Sialidase‐treated samples showed a microheterogeneous banding pattern which consisted of two major and several additional minor components with isoelectric points between pH 5.0 and 5.3. A population study of 200 unrelated individuals from southern Germany revealed no genetic variation. In a clinical investigation, however, we found a unique banding pattern in a female patient suffering from chronic obstructive pulmonary disease. In comparison with the monomorphic normal type the detected variant phenotype shows two additional bands that have lower intensities and are located cathodically to their major bands. Inheritance of the deficient IEF variant “ACT Bochum” was confirmed by a family study. To our knowledge this is the first genetic ACT mutant to be observed at the protein level.
Isoelectric focusing and immunoblotting of the platelet membrane glycoprotein complex IIb. IIIA following urea solubilization
Authors: Gianazza Elisabetha , Frattini Roberta , Michelagnoli Silvia , Cassinotti Michela , Sirtori Cesare R.
Published: May 2005

Effective solubilization of the major platelet membrane component, the glycoprotein IIb. IIIa complex, can be achieved with 8 M urea. By avoiding nonionic detergents in the separation medium it is possible to obtain clear immunoblot patterns without interference from the isoelectric focusing matrix. Upon running on a pH 4.25–5.25 immobilized pH gradient, immunoreactive bands corresponding to the nonreduced IIb.IIIa complex stain between pH 4.5 and 5.0. The method appears of significant potential utility in evaluating glycoprotein IIb.IIIa polymorphisms under different clinical conditions.
Genetic study of orosomucoid by isoelectric focusing and immunoprinting in patients with carcinoma
Authors: Mittermüller Johann , Weidinger Sebastian
Published: May 2005

The carbohydrate moiety of the orosomucoid (ORM) molecule shows microheterogeneity [1] and the pteridine‐containing variant seems to be tumor‐specific [2–4]. However, there also exists a genetic (protein‐related) polymorphism coded by the ORM1 and ORM2 loci on chromosome 9 [5, 6]. To investigate the relationship between ORM1 gene products and the development of carcinoma, we analyzed the ORM1 phenotypes of desialated sera 125 patients with carcinoma. The allele frequencies were estimated for ORM1*F1 0.556, ORM1*F2 0.012 and ORM1*S 0.432. In comparison to healthy individuals from the same geographical area [6] the ORM1 S phenotypes are significantly more frequent in carcinoma patients. The patients' sera frequently showed additional ORM‐positive proteins which focused slightly cathodically to the ORM 2 A band. These proteins may represent posttranslational modifications of the ORM1*S allele product. Whether these modifications are tumor‐specific and related to the carbohydrate moiety of the molecule must be confirmed in further studies.