bluuebunny/crossref_metadata_2025 huggingface dataset:
The molecular chaperones HSP28, GRP78, endoplasmin, and calnexin exhibit strikingly different levels in quiescent keratinocytes as compared to their proliferating normal and transformed counterparts: cDNA cloning and expression of calnexin
Authors: Honoré Bent , Rasmussen Hanne H. , Celis Ariana , Leffers Henrik , Madsen Peder , Celis Julio E.
Published: May 2005

We have identified nine molecular chaperones in human keratinocytes by one or a combination of three methods: (i) reaction with antibodies raised against the purified proteins, (ii) microsequencing of two‐dimensional (2‐D) gel purified proteins, or (iii), by cloning of the cDNA and expression of its encoded protein in transformed human amnion cells using the vaccinia virus expression system. The expression levels of each of the molecular chaperones were analyzed in quiescent, normal proliferating, and simian virus SV40 transformed K14 keratinocytes by cutting the corresponding protein spots from dried 2‐D gels and counting the radioactivity by liquid scintillation. The most striking observation was the strong up‐regulation (936%) of the small heat shock protein HSP28 in the quiescent keratinocytes, a fact that is in line with recent data indicating that the murine homologue (HSP25) may act as a growth inhibitor. Several chaperones that localize to the endoplasmic reticulum and that are involved in the secretory pathway (GRP78, GRP78v, endoplasmin, and calnexin) were expressed at approximately similar levels in normal proliferating and K14 keratinocytes but were down‐regulated by 50% or more in the quiescent cells, implying that these cells may possess an impaired ability to secrete certain proteins. Both GRP78 and endoplasmin genes have similar sequences in the promoter regions, suggesting that they may be partly co‐regulated at the transcriptional level (McCauliffe et al., J. Biol. Chem. 1992, 267, 2557–2562). Whether this also applies to the calnexin promoter region must await cloning of the gene which is now possible with the human cDNA reported here. Only three chaperones were up‐regulated in K14 (HSP60, HSC70, and HSP90), and their increased expression may be related to the transformed phenotype.
Complex protein composition of isolated focal adhesions: A two‐dimensional gel and database analysis
Authors: Niederreiter Markus , Gimona Mario , Streichsbier Franz , Celis Julio E. , Small J. Victor
Published: May 2005

Current ideas about the composition of the focal adhesion complexes in cultured cells are based mainly on indirect immunocytochemical data. We here report a two‐dimensional (2‐D) gel electrophoresis analysis of the focal adhesion associated‐structures that remain in the growth substrate after removal of cells by mechanical shearing. Many proteins additional to the known adhesion proteins, and in higher abundance, could be identified. Using selective extraction procedures, employing detergent or gelsolin, these could be classified as either membrane‐associated, actin‐associated or both. Cross correlation of these polypeptode patterns with a 2‐D gel database allowed identification of some proteins, not previously considered as resident of focal adhesions. The data point to a more complex make up of focal adhesions than formerly supposed.
Reference points for comparisons of two‐dimensional maps of proteins from different human cell types defined in a pH scale where isoelectric points correlate with polypeptide compositions
Authors: Bjellqvist Bengt , Basse Bodil , Olsen Eydfinnur , Celis Julio E.
Published: May 2005

A highly reproducible, commercial and nonlinear, wide‐range immobilized pH gradient (IPG) was used to generate two‐dimensional (2‐D) gel maps of [35S]methionine‐labeled proteins from noncultured, unfractionated normal human epidermal keratinocytes. Forty one proteins, common to most human cell types and recorded in the human keratinocyte 2‐D gel protein database were identified in the 2‐D gel maps and their isoelectric points (pI) were determined using narrow‐range IPGs. The latter established a pH scale that allowed comparisons between 2‐D gel maps generated either with other IPGs in the first dimension or with different human protein samples. Of the 41 proteins identified, a subset of 18 was defined as suitable to evaluate the correlation between calculated and experimental pI values for polypeptides with known composition. The variance calculated for the discrepancies between calculated and experimental pI values for these proteins was 0.001 pH units. Comparison of the values by the t‐test for dependent samples (paired test) gave a p‐level of 0.49, indicating that there is no significant difference between the calculated and experimental pI values. The precision of the calculated values depended on the buffer capacity of the proteins, and on average, it improved with increased buffer capacity. As shown here, the widely available information on protein sequences cannot, a priori, be assumed to be sufficient for calculating pI values because post‐translational modifications, in particular N‐terminal blockage, pose a major problem. Of the 36 proteins analyzed in this study, 18–20 were found to be N‐terminally blocked and of these only 6 were indicated as such in databases. The probability of N‐terminal blockage depended on the nature of the N‐terminal group. Twenty six of the preteins had either M, S or A as N‐terminal amino acids and of these 17–19 were blocked. Only 1 in 10 proteins containing other N‐terminal groups were blocked.
Distinction of weakly homologous cDNA amplificates by single‐strand conformation polymorphism analysis: Application to guanylyl cyclase isozymes
Authors: Krause Günter , David Sigrid , Büchler Wolfgang , Wehling Martin , Gerzer Rupert
Published: May 2005

By use of the polymerase chain reaction (PCR), uniform amplification products of 225 to 240 bp length were obtained from five cDNA clones representing different types of guanylyl cyclases. These short DNA double strands were differentiated by single‐strand conformation polymorphism (SSCP), using polyacrylamide gel electrophoresis with the Pharmacia Phast‐System. Following heat denaturation, the samples were separated on native polyacrylamide gels at different running temperatures. Nucleic acids on the gel were detected by an automated silver stain procedure. Using 7.5% homogeneous or 4–15% gradient polyacrylamide gels at a temperature of 12°C, single‐strand conformations of amplificates, representing three different particulate guanylyl cyclases and the two subunits of soluble guanylyl cyclase, were differentiated. The characteristic banding patterns resulting from dissimilar migration of the single‐strand conformations were assigned to different guanylyl cyclase types. For the enzyme family of guanylyl cyclases, the feasibility of a combined PCR and electrophoresis approach for analyzing the expression of related genes was demonstrated. This application of the PCR‐SSCP technique provided a rapid and sensitive tool for the characterization of PCR products obtained with a common primer pair and suggested its use for investigating the tissue distribution of gene expression within a class of homologous proteins.
Classification of wheat varieties by isoelectric focusing patterns of gliadins and neural network
Authors: Søndergaard Ib , Jensen Kirsten , Krath Britta Naimi
Published: May 2005

Classification of wheat varieties, using isoelectric focusing patterns of the gliadins, image processing and neural networks, is described. The method was compared to a statistical classification method, discriminant analysis. The isoelectric point and the area of each band were calculated by image processing. Different methods of presenting the electrophoretic patterns to the neural network were studied. The most effective method was transformation of the electrophoretic pattern to a small (11 × 47 pixels) representation of the original digitized image, which was presented to the neural network as a vector. The neural network was trained with a number of patterns and tested with new patterns from different electrophoretic runs of the same wheat varieties. In this study we used ten different wheat varieties and the neural network was able to classify 95.5% of the patterns correctly. The statistical classification method classified the same data set 91.8% correctly. We conclude that both the neural network and discriminant analysis were able to classify the patterns correctly with a high degree of certainty. The patterns that were misclassified were indistinguishable by visual inspection.
Rapid method for isolating detergent‐insoluble outer membrane proteins from Pseudomonas aeruginosa
Authors: Ravaoarinoro Madeleine , Ciurli Cristina , Toma Emil , Morisset Richard
Published: May 2005

The present study compares two techniques for isolating outer membrane proteins (OMPs) of Pseudomonas aeruginosa, Method A ‐ selective solubilization with sodium lauryl sarcosinate, and Method B ‐ isopycnic sucrose gradient centrifugation, using three criteria: the amount of proteins obtained, polypeptide patterns after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and their practical aspects. Method A appears to be superior to Method B. It yields a higher outer membrane protein content and a similar polypeptide pattern as Method B, but is more rapid and less cumbersome.
A transient entanglement coupling mechanism for DNA separation by capillary electrophoresis in ultradilute polymer solutions
Authors: Barron Annelise E. , Blanch Harvey W. , Soane David S.
Published: May 2005

Using capillary electrophoresis, large DNA molecules (2.0–23.1 kbp) may be rapidly separated in ultradilute polymer solutions (< 0.002% w/w) under, a high‐voltage, steady field (265 V/cm). At this polymer concentration, the separation mechanism appears to be significantly different from that postulated to occur in crosslinked gels. Based on experimental results obtained with DNA restriction fragments and with negatively charged latex microspheres, we conclude that the Ogston and reptation models typically used to describe gel electrophoresis are not appropriate for DNA separations in such dilute polymer solutions. Electrophoresis experiments employing solutions of both small and large hydroxyethyl cellulose polymers highlight the importance of polymer length and concentration for the optimum resolution of DNA fragments varying in size from 72 bp to 23.1 kbp. A transient entanglement coupling mechanism for DNA separation in dilute polymer solutions is developed, which suggests that there is no a priori upper size limit to DNA that can be separated by capillary electrophoresis in a constant field.
Capillary zone electrophoresis of derivatized acidic monosaccharides
Authors: Mechref Yehia , El Rassi Ziad
Published: May 2005

A new and specific precolumn derivatization reaction for acidic monosaccharides was introduced and evaluated in the separation and sensitive detection of carbohydrates by capillary electrophoresis. The derivatization reaction involved the attachment of sulfanilic acid (a UV absorbing tag) or 7‐aminonaphthalene‐1,3‐disulfonic acid (a UV absorbing and fluorescing tag) via a condensation reaction between the amino group of the derivatizing agent and the carboxyl group of the sugar in the presence of a water‐soluble carbodiimide. The derivatization reaction replaced the weak carboxylic acid of the sugar by a strong sulfonic acid, which is fully ionized at all pH. This allowed the electrophoresis of the sugar derivatives over a wide pH range and permitted the determination of acidic carbohydrates at very low femtomole levels by UV and fluorescence detection.
A new method for the detection of proteolytic activity in Pseudomonas lundensis after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Lundy Fionnuala T. , Magee Anthony C. , Blair Ian S. , McDowell David A.
Published: May 2005

A new method for the visualization of proteolytic activity in cell culture supernatant from Pseudomonas lundensis after sodium dodecyl sulfate (SDS) – gel electrophoresis is described. Following conventional electrophoresis, the gel is washed in a methanol‐containing buffer to facilitate partial removal of SDS. After incubation with 0.5% casein the gel is stained for protein with Coomassie Brilliant Blue R‐250. Bands with proteolytic activity appear as clear areas in the gel against a blue‐stained background. Molecular weight standards electrophoresed in the same gel stain more intensely than the background and allow determination of the molecular weights of the proteolytic components. The sensitivity of post‐electrophoretic reactivation in SDS‐gels was determined using trypsin as standard. A slight modification of the technique allowed detection of proteolytic activity in nondenaturing and in isoelectric focusing gels.
Surface‐charge reversed capillary zone electrophoresis of inorganic and organic anions
Authors: Oefner Peter J.
Published: May 2005

Anions commonly found in biological fluids such as chloride, citrate, phosphate, bicarbonate, lactate, aspartate and glutamate, were determined within 60 s by means of capillary zone electrophoresis and indirect UV detection using 10 mM of chromate, pH 8.0, as both carrier electrolyte and chromophore. The lower mass detection limit was 0.5 pmol. In order to avoid a gradual shift of the pH and, in consequence, drifting ionic mobilities due to the formation of hydroxyl ions at the cathode, 1 mM of 5,5‐diethylbarbiturate was added to the background electrolyte. Moreover, fused silica capillaries were coated with a 0.0003% solution of hexadimethrine bromide for two minutes between runs to reverse electroendosmotic flow which otherwise would counteract the electrophoretic migration of the anions and, hence, increase time of analysis. It is concluded that charge‐reversed capillary zone electrophoresis in concert with indirect UV detection is superior to ion chromatography and conductivity detection in the determination of inorganic and organic anions in biological fluids both with regard to selectivity, sensitivity and speed of analysis

Presented in part at the International Ion Chromatography Symposium, Linz, Austria, September 21–24, 1992

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Detection of platinum species in plant material
Authors: Messerschmidt Jürgen , Alt Friedrich , Tölg Günther
Published: May 2005

Model experiments for the detection of platinum species in extracts from native and platinum‐treated grass cultivations are described. The procedural steps are cultivation of the grass samples, extraction and concentration of the platinum species by ultrafiltration and freeze‐drying, preparative separation of the species by gel chromatography followed by isotachophoresis, and sequential analytical detection of the separated platinum species by adsorptive voltammetry. After isotachophoresis, sharp peaks of platinum species could be detected. In the native grass extract only one platinum species (160–200 kDa) was found. In the platinum‐treated grass extracts several platinum species were observed in the molecular mass range from 1 to > 1000 kDa. By an extremely sensitive platinum determination method (adsorptive voltammetry; detection limit, 2 pg Pt abs.) it was possible to detect platinum even in stained protein bands from horizontal gel electrophoresis of platinum containing fractions obtained after isotachophoresis.
Detection of immunoglobulin G glycosylation changes in patients with rheumatoid arthritis by means of isoelectric focusing and lectin‐affinoblotting
Authors: Hänsler Marion , Kötz Kerstin , Häntzschel Holm
Published: May 2005

Patients with rheumatoid arthritis have a reduced prevalence of immunoglobulin G (IgG) oligosaccharide chains terminating in galactose, thus exposing N‐acetylglucosamine. We analyzed IgG glycosylation in patients with rheumatoid arthritis, patients with early synovitis, and in controls by means of isoelectric focusing and lectin‐affinoblotting. The ratio of N‐terminal N‐acetylglucosamine and galactose was determined using specific biotin‐labeled lectins. The IgG glycosylation state may well be of clinical value in the differential diagnosis of patients presenting with early synovitis.
Mineral‐bound noncollagenous proteins in archaeological human skeletons
Authors: Freundorfer Stephan , Grupe Gisela , Weickmann Dirk
Published: May 2005

Archaeometric approaches to archaelogical human bone also include the extraction, identification and molecular analysis of surviving bone proteins. Due to its abundance as a matrix protein, most studies focus on collagen (e.g. radiocarbon dating). Also, a variety of serum proteins are detectable in excavated skeletons. Very limited knowledge still exists on mineral‐bound noncollagenous bone proteins from ancient bones because, in the mature tissue, they occur in trace amounts only. Moreover, post‐mortem decomposition is likely to change characteristic features of the molecules. Due to their suggested role as growth and developmental factors, identification and quantification of such proteins should be valuable for both physical anthropology and epidemiology. We present a valid method for the detection of small amounts of surviving mineral‐bound noncollagenous proteins in excavated human bones up to 7500 years of age.
Theory of the mobility‐shift assay of nonspecific protein‐DNA complexes governed by conditional probabilities: The HU:DNA complex
Authors: Cann John R. , Pfenninger Oswald , Pettijohn David E.
Published: May 2005

Complexes of an 88 bp DNA and the HU protein were studied by both experimental and theoretical electrophoretic mobility‐shift analyses. Experimental analysis defined the stoichiometry of binding and estimated an apparent intrinsic dissociation constant (Kd = 1 to 3 × 10−7 M) for the HU:DNA complexes. The theory of conditional probabilities was applied to the binding of HU to DNA in order to fix the initial equilibrium composition of mixtures to be assayed theoretically by the mobility‐shift procedure. Electrophoretic mobility‐shift patterns were obtained by numerical solution of a set of simultaneous transport‐reaction equations, in which the chemical kinetic term is formulated in terms of dissociation of the different DNA:HU complexes in gel cages. The computed patterns simulated the experimental patterns describing the titration of a fixed concentration of an 88 bp DNA fragment with dimeric HU. These insightful results provide guidelines for interpretation of the electrophoretic behavior of systems in which a ligand binds nonspecifically to DNA. In particular, the narrow unresolved zone observed both experimentally and theoretically beyond 50–60% saturation is a reaction zone characteristic of noncooperative ligand‐binding governed by conditional probabilities. The discrepancy between the theoretically assigned and experimental values of the intrinsic binding constant is attributed to an HU‐induced change in the conformation of DNA.
A silver stain protocol for proteins yielding high resolution and transparent background in sodium dodecyl sulfate‐polyacrylamide gels
Authors: Swain Marianne , Ross Neil W.
Published: May 2005

A simple, sensitive silver staining method for sodium dodecyl sulfate polyacrylamide gel electrophoresis has been developed which yields high resolution of proteins with a transparent background. The method includes the incorporation of a water wash after a short fixation step in ethanol/acetic acid and prior to glutaraldehyde cross‐linking, which appears to be necessary for the high resolution staining of protein bands and the low background staining.
Dynamics of peak dispersion in capillary zone electrophoresis including wall adsorption I. Theoretical model and results of simulation
Authors: Gaš Bohuslav , Štědrý Milan , Rizzi Andreas , Kenndler Ernst
Published: May 2005

A mathematical model is described for the simulation of peak profiles in capillary zone electrophoresis taking wall adsorption into account. It is based on such physico‐chemical relations as mass balance equations, sorption rate equations and appropriate boundary conditions. The numerical solution of the model is carried out, allowing the depiction of the concentration profile of the sample in the capillary as a result of the dynamics of the processes involved, which depend on a number of input parameters: rate constants, adsorption isotherms, column dimensions, field strength etc. Four cases are discussed in detail, namely those with either slow or fast adsorption kinetics and linear or nonlinear isotherms.
Application of dual internal standards for precise sizing of polymerase chain reaction products using capillary electrophoresis
Authors: Butler John M. , McCord Bruce R. , Jung Janet M. , Lee James A. , Budowle Bruce , Allen Ralph O.
Published: May 2005

Capillary electrophoresis (CE) is an analytical technique which provides rapid, high resolution analysis of amplified DNA fragments produced by the polymerase chain reaction (PCR). In this study, two internal standards are used as size markers to bracket und precisely size PCR products. The technique is applied to typing PCR products from the short tandem repeat locus HUMTH01. HUMTH01 consists of five to seven major alleles in the size range of 179–203 bp, with each allele four bp apart. Using this genetic marker, a population containing 97 individuals was examined with both polyacrylamide gel electrophoresis and CE. Identical genotypes were obtained with both techniques demonstrating the reliability of CE in DNA typing applications. The DNA analysis took place in sets of 10 with a calibration of the CE being performed between each set of samples. For the 97 samples examined, the pooled standard deviation was 0.3 bp. The observed genotype frequencies determined from the sample set did not deviate significantly from Hardy‐Weinberg expectations. From these CE results, we conclude that HUMTH01 PCR products can be accurately and precisely sized by capillary electrophoresis using the method described.
Capillary electrophoresis of rat liver microsomes in polymer solutions
Authors: Radko Sergey P. , Sokoloff Alexander V. , Garner Mark M. , Chrambach Andreas
Published: May 2005

Rat liver microsome components were separated by capillary zone eletrophoresis in buffers containing substituted agarose, agarose crosslinked polyacrylamide, polyacrylamide, polyethylene glycol and dextran of various molecular weights. The best resolution of the components was obtained with polymers of 10–21 nm geometric mean radius. Both the crude and the purified preparations of microsomes exhibit a single major peak. In a Tris‐borate‐EDTA (TBE) buffer, containing polyacrylamide of 5 × 106 molecular weight, it has a retardation coefficient, KR, of 0.77 × 0.02. Translation of the KR value to geometric mean radii, R, on the basis of the standard curve applicable to polymer solutions, KR vs. R, with polystyrene carboxylate size standards in both dextran and polyacrylamide solution allows one to estimate a value of R as 13–16 nm for the major microsome component. The value is smaller than expected from electron microscopic measurements (100–250 nm), possibly due to the chemical and geometric differences between microsomes and the polystyrene particles used as size standards. The crude preparation also contains a minor component which is smaller and less charged than the major component. Another component, apparently very much larger than the major one, is seen in TBE buffer but not in a potassium‐N‐(2‐hydroxyethyl)piperazine‐2′‐(2‐ethanesulfonic acid) buffer and is therefore thought to be an artifact of interaction with borate. After a short incubation under conditions promoting delayed microsome fusion, i.e. in presence of GTP and Mg++ and in the absense of polyethylene glycol, the electrophoretic pattern changes dramatically: it now exhibits five unretarded highly mobile and, therefore, presumbly large components in addition to the two original retarded components of the microsome and a less highly charged species similar in KR to the smaller of the original two components.
Capillary electrophoresis of human serum proteins and apolipoproteins
Authors: Lehmann Rainer , Liebich Hartmut , Grübler Gerald , Voelter Wolfgang
Published: May 2005

Capillary electrophoresis (CE) analysis of human serum proteins and apolipoproteins is described and compared with sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis and cellulose acetate membrane electrophoresis. Under optimized CE conditions apolipoproteins I could be determined directly in the serum and quantitated with a linear correlation between peak area and concentration up to a concentration of 100 mg/dL.
Visualization of barley β‐glucan degrading isozymes after gel isoelectric focusing
Authors: Menteur Sophie , Jestin Louis , Risacher Thierry , Branlard Gérard
Published: May 2005

A simple method to study the polymorphism of barley (Hordeum vulgare L.) β‐(1–3, 1–4)‐glucanases (specific enzymes of barley β‐glucan hydrolysis) is described. Proteins of a crude extract of germinated barley kernels were separated in an immobilized pH gradient in two pH ranges (pH 3–10.5 and pH 4–7). β‐glucanases were visualized by contact printing with a polyacrylamide gel containing β‐glucan or lichenan. Patterns of β‐glucanases were revealed by staining with Congo Red with resultant clear zones on a stained background. Various conditions of germination, extraction and visualization were investigated. New isozyme bands could be detected and their nature and origin discussed.
Transient bisalbuminemia: Separation by isoelectric focusing of human albumin fractions linked to different numbers of benzylpenicilloyl groups
Authors: Rocha Jorge , Bohner Jürgen , Kömpf Jost
Published: May 2005

The behavior of modified human serum albumin (HSA) from penicillin‐treated patients and in vitro conjugated samples was studied by isoelectric focusing in the absence and presence of 8 M urea. It is shown that samples classified as bisalbuminemic after separation under native conditions display a spectrum of several major bands with decreasing pIs when focusing is done in the presence of 8 M urea. In this spectrum, the distances between two consecutive major bands are as expected from a 2‐unit charge difference. Since this result is compatible with the blockage of a lysine residue with a benzylpenicilloyl (BPO) group, each major band can be interpreted as representing an HSA fraction covalently linked to different numbers of benzylpenicilloyl groups. The separation methods presented therefore seem to constitute simple and reliable tools for a detailed assessment of the extent of BPO covalent binding to HSA both in vivo and in vitro.
Two‐dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome
Authors: Klose Joachim , Kobalz Ursula
Published: May 2005

The two‐dimensional electrophoresis (2‐DE) technique developed by Klose in 1975 (Humangenetik 1975, 26, 211–234), independently of the technique developed by O'Farrell (J. Biol. Chem. 1975, 250, 4007–4021), has been revised in our laboratory and an updated protocol is presented. This protocol is the result of our experience in using this method since its introduction. Many modifications and suggestions found in the literature were also tested and then integrated into our original method if advantageous. Gel and buffer composition, size of gels, use of stacking gels or not, necessity of isoelectric focusing (IEF) gel incubation, freezing of IEF gels or immediate use, carrier ampholytes versus Immobilines, regulation of electric current, conditions for staining and drying the gels – these and other problems were the subject of our concern. Among the technical details and special equipment which constitute our 2‐DE method presented here, a few features are of particular significance: (i) sample loading onto the acid side of the IEF gel with the result that both acidic and basic proteins are well resolved in the same gel; (ii) use of large (46 × 30 cm) gels to achieve high resolution, but without the need of unusually large, flat gel equipment; (iii) preparation of ready‐made gel solutions which can be stored frozen, a prerequisite, among others, for high reproducibility. Using the 2‐DE method described we demonstrate that protein patterns revealing more than 10 000 polypeptide spots can be obtained from mouse tissues. This is by far the highest resolution so far reported in the literature for 2‐DE of complex protein mixtures. The 2‐DE patterns were of high quality with regard to spot shape and background. The reproducibility of the protein patterns is demonstrated and shown to be thoroughly satisfactory. An example is given to show how effectively 2‐DE of high resolution and reproducibility can be used to study the genetic variability of proteins in an interspecific mouse backcross (Mus musculus × Mus spretus) established by the European Backcross Collaborative Group for mapping the mouse genome. We outline our opinion that the structural analysis of the human genome, currently pursued most intensively on a worldwide scale, should be accompanied by a functional analysis of the genome that starts from the proteins of the organism.
Two‐dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimensin (IPG‐Dalt): The state of the art and the controversy of vertical versus horizontal systems
Authors: Görg Angelika , Boguth GÜNther , Obermaier Christian , Posch Anton , Weiss Walter
Published: May 2005

After having established the basic protocol of two‐dimensional electrophoresis with immobilized pH gradients in the first dimension (IPG‐Dalt) in 1988 (A. Görg et al., Electrophoresis 1988, 9, 531–546), some critical parameters of the actual IPG‐Dalt protocols as well as the results obtained with horizontal and vertical second‐dimensional sodium dodecyl sulfate‐electrophoresis are demonstrated and discussed.
Sequence analysis of wheat grain allergens separated by two‐dimensional electrophoresis with immobilized pH gradients
Authors: Posch Anton , Weiss Walter , Wheeler Colin , Dunn Michael J. , Görg Angelika
Published: May 2005

Micropreparative two‐dimensional (2‐D) gel electrophoresis with immobilized pH gradients (4–8) in the first dimension (IPG‐DALT) was optimized for the separation of salt‐soluble wheat grain proteins associated with bakers' asthma disease. The resolved polypeptides were electroblotted onto a polyvinylidene difluoride (PVDF) membrane and incubated with the pooled sera from four asthmatic bakers. Bound IgE was demonstrated by alkaline phosphatase conjugated anti‐human IgE. Major IgE binding was detected in the 27 kDa, 37 kDa and, to a lesser extent, in the 14–18 kDa area of the 2‐D immunoblots, respectively. Since the main purpose of our study was to determine the N‐terminal amino acid sequences of the major wheat grain allergens, N‐terminal sequencing was performed for six out of a total of eleven major allergens located in the 27 kDa area, for one out of two 37 kDa allergens, and for two out of four 14–18 kDa allergens. Our results revealed that two of the 27 kDa polypeptides are clearly related to several Acyl‐CoA oxidase variants of barley and rice, whereas no significant homologies were found for the remaining four 27 kDa allergens analyzed. The N‐terminus of the 37 kDa allergen appeared to be blocked so that no sequence information was obtained, while the two 14–18 kDa allergens analyzed were identified as members of the wheat α‐amylase‐inhibitor family.
Inside SWISS‐2DPAGE database
Authors: Sanchez Jean‐Charles , Appel Ron D. , Golaz Olivier , Pasquali Christian , Ravier Florence , Bairoch Amos , Hochstrasser Denis F.
Published: May 2005

Several two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) databases have been established and updated for more than 15 years. Only recently have developments of computer networks and high‐speed transfer protocols provided the required tools for sharing comprehensive and hypermedia 2‐D PAGE databases. This publication describes the SWISS‐2DPAGE database structure. Proteins present in samples of human tissue, cells, cell lines and body fluids are assembled and described in an accessible uniform format. SWISS‐2DPAGE can be freely accessed through the World‐Wide Web (WWW) network on the ExPASy molecular biology server.
Rapid detection of the main human plasma glycoproteins by two‐dimensional polyacrylamide gel electrophoresis lectin affinoblotting
Authors: Golaz Olivier , Gravel Patricia , Walzer Claude , Turler Hans , Balant Luc , Hochstrasser Denis F.
Published: May 2005

Glycoprotein modifications in the glycan moiety can occur in diseases such as cancers, inflammatory processes and alcoholism. We combined high‐resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) with lectin affinoblotting in order to establish the normal human plasma glycoprotein map. Human plasma proteins were separated by mini 2‐D PAGE (7 × 9 cm), transferred onto polyvinylidene difluoride membranes and incubated with biotinylated lectins. We focused our study on lectins binding sialic acid and galactose residues. Known plasma glycoproteins such as αl‐antitrypsin, αl‐antichymotrypsin, α2‐HS glycoprotein, αl‐acid glycoprotein, haptoglobin β‐chain and transferrin were easily detected in ng amounts. This protocol was adequate to establish a normal plasma glycoprotein map and will allow the study of glycoproteins in diseases.
Effects of cyclosporine A on the rat liver and kidney protein pattern, and the influence of vitamin E and C coadministration
Authors: Benito Begoña , Wahl Daniel , Steudel Nicola , Cordier André , Steiner Sandra
Published: May 2005

The effects of cyclosporine A (CsA), a potent immunosuppressive drug, were examined in rat liver and kidney samples using two‐dimensinal electrophoretic protein analysis. Of a total of 370 liver and 336 kidney spots analyzed, 8% (29 spots) and 6% (19 spots), respectively, showed a significant drug‐induced change (p < 0.01), which was predominantly reflected in increased protein abundance (62% and 74% of the changes, respectively). Of the 48 proteins changed in either organ, 14 were most probably common to both tissues and one of these was significantly increased in both the liver and the kidney. Most of the other 13 showed similar trends (either increases or decreases) in both organs. However, the most striking drug effect seen in this study concerned an unindentified protein present only in the kidney, which completely disappeared upon CsA treatment. It was also investigated whether the drug‐induced changes could be prevented by the coadministration of the radical scavengers vitamin E and C with CsA. Spots changed by the administration of the drug were classified according to three different categories, based on their response profiles in rats treated with CsA in combination with the vitamins: (i) spots which were changed by CsA as well as by CsA in combination with the vitamins (12 liver and 4 kidney spots), (ii) spots which were changed by CsA and showed an additional increase of this change by CsA plus the vitamins (no liver and 4 kidney spots), and (iii) spots which were changed by CsA but not by CsA in combination with the vitamins (8 liver and 6 kidney spots). These results showed that in both organs the vitamins were able to prevent around 30% of the effects caused by CsA, and that two‐dimensional gel electrophoresis is an excellent tool to demonstrate such drug interactions at the molecular level.
Microheterogeneity of mouse antidextran monoclonal antibodies
Authors: Mimura Yusuke , Kabat Elvin A. , Tanaka Tatehiko , Fujimoto Masanori , Takeo Kazusuke , Nakamura Kazuyuki
Published: May 2005

Mouse antidextran monoclonal antibodies showed microheterogeneity which was analyzed by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). Not only the heavy (H) chains but also the light (L) chains were heter ogeneous in terms of isoelectric point (pI). The higher the pI, the more prominent the H chain spots. To demonstrate the cause of the microheterogeneity an IgG1 monoclonal antibody (mAb 35.8.2H) was examined especially for involvement of the sugar moiety in the microheterogeneity. The glycosylated region was determined in the Fc portion from serine 239 to methionine 309 by a glycan detection method using mild periodate oxidation, which confirms that the sugar chain is attached to the conserved glycosylation site of asparagine 297. However, charge heterogeneity of the H chain was not entirely attributed to the Fc because the papain digest of the antibody was separated into two Fc spots, a few Fd spots and two L chain spots by 2‐D PAGE. This indicates that factors other than the sugar moiety are responsible for charge heterogeneity of IgG monoclonal antibody. On the other hand, the H chain isoforms of lower pI were shown to be more susceptible to V8 protease by peptide mapping. This result strongly suggests the occurrence of deamidation at glutamine or asparagine residues.
Distance indices in a comparison between the A, D, I and R genomes of the Triticeae tribe
Authors: Zivy Michel , Madidi Saïd El , Thiellement Hervé
Published: May 2005

Two‐dimensional gel electrophoresis of shoot proteins was used to study the relationships between Triticum, Secal, and Hordeum. A high level of polymorphism was found among the 1275 spots scored: only 198 spots were found common to all. But, under the hypothesis that only allelic variations were observed, the mean number of alleles per locus was only two. Phenograms were built from different distance indices. All of them showed Triticum genomes A and D close to each other, Hordeum, far from the Triticum cluster, and Secale at an intermediate position. A discussion on the use of various distance indices is presented.
Electrophoretic, chromatographic and immunological studies of human urinary proteins
Authors: Büeler Martin R. , Wiederkehr Felix , Vonderschmitt Dieter J.
Published: May 2005

Urinary proteins from both sexes were analyzed by high resolution two‐dimensional gel electrophoresis (2‐DE). For well reproducible 2‐DE patterns, the samples were concentrated and desalted in one step by vacuum dialysis. A reference map for urine proteins was established by the analysis of urine from 10 healthy persons. Proteins in urine that share immunogenicity with serum proteins were identified by use of antibody to whole human‐serum protein in an affinity‐column fractionation of urine and differential analysis of the adsorbed (serum component) and unadsorbed (non‐serum component) fractions. For identification of individual proteins, coelectrophoresis, immunoblotting and affinity chromatography with corresponding antibodies were used. Proteins identified in the map, besides known serum proteins, included: the subunit of Tamm‐Horsefall protein, the secretory component of IgA, constant breakdown products of αl‐antitrypsin and retinol‐binding protein, the five isoforms of the β chain of human chorionic gonadotropin and the subunit of prostatic acid phosphatase. In addition, we could demonstrate three proteins which are markedly pronounced in female urine, especially pregnant women. To get more information about the native properties of various urinary proteins, they were separated into four main peaks according to their sizes using fast protein liquid chromatography equipment. Possible interpolypeptide disulfide bonds were studied using a nonreducing 2‐DE system. 2‐DE in combination with other methods seems to be a valuable tool for the characterization of urinary proteins in defined renal or extra‐renal diseases. An example is given by analyzing the immune complexes from seven patients with a urinary tract infection.
Electrophopretic mobility of soft particles
Authors: Ohshima Hiroyuki
Published: May 2005

A recent theory of electrophoresis of “soft” particles, i.e., polyelectrolytecoated colloidal particles (Ohshima, J. Colloid Interface Sci. 1994, 163, 474–483), is reviewed. This theory unites two different electrophoresis theories for rigid colloidal spherical particles and for spherical polyelectrolytes. An example is given of the analysis of electrophoretic mobility data on hydrogel‐layer‐coated latex particles.
A computer model for time‐based simulation of electrophoresis systems with freely defined initial and boundary conditions
Authors: Schafer‐Nielsen Claus
Published: May 2005

A PC‐based program has been developed that allows the user to perform one‐dimensional computer simulations of electrophoresis systems with freely definable initial‐ and boundary conditions. The program can handle n constituents with n pK‐values and calculates constituent concentrations and derived parameters as a function of time. Results are displayed graphically on screen and are stored as data files for graphical hard‐copy processing.
Sensitive quantification of proteins by electrophoresis in gels by use of chemiluminescence
Authors: Vesterberg Olof , Acevedo Fernando , Bayard Cecilia
Published: May 2005

For protein quantitation in gels or blotting membranes, chemiluminescence (CL) offers the advantages of a substantial improvement of detection limits. Easy‐to‐use CL‐chemicals and specific probes such as antibodies conjugated to enzymes, e.g. alkaline phosphatase (AP) may be used in combination with a newly developed luminometer. CL was found to have low detection limits and a linear relation between relative light units (RLU) and the concentration of the antibody enzyme complex present over a wide concentration range. Measurements of the immunoglobulin IgE in dot blots and in blots after sodium dodecyl sulfate (SDS) electrophoresis under nonreducing conditions in agarose gels are described.
Electrophoretic size separation of proteins treated with sodium dodecyl sulfate in 1% agarose gels
Authors: Acevedo Fernando , Marín Viviana , Wasserman Moisés
Published: May 2005

Separation of proteins treated with sodium dodecyl sulfate (SDS) according to molecular size was achieved by discontinuous electrophoresis in vertical low‐concentration agarose gels. A linear relationship was found between the migration distance and the square root of the molecular weight. This holds for proteins in the range of 7–200 kDa separated in 1.25% w/v agarose gel slabs (7 × 7 × 0.15 cm) with 0.1% w/v SDS and sulfate as leading ion. The linear regression coefficient was 0.998. The molecular weight and charge of coions influenced the separation. Small ions with low pKa values were found suitable as coions. The migration distance of proteins treated with SDS varied linearly with the agarose concentration of the gel. The agarose type and quality affected the resolution of the SDS‐protein bands. We conclude that agarose gels can substitute polyacrylamide gels for the separation of proteins treated with SDS. A homogeneous agarose gel at a concentration of about 1% is a nonsieving support for electrophoresis. Therefore, the separation described here cannot be explained by the pore size of the gel. The results suggest that the separation is mainly due to the relative migration velocities of the coion and the proteins treated with SDS.
Rapid typing of variable number of tandem repeat locus in the human apolipoprotein B gene for DNA diagnosis of heart disease by polymerase chain reaction and capillary electrophoresis
Authors: Baba Yoshinobu , Tomisaki Riyo , Sumita Chinuyo , Morimoto Izumi , Sugita Sanae , Tsuhako Mitsutomo , Miki Tetsuro , Ogihara Toshio
Published: May 2005

The apolipoprotein B (apoB) variable number of tandem repeat (VNTR) alleles containing larger repeat units is a risk factor for coronary heart disease. Capillary electrophoresis (CE) in entangled polymer solution was applied to the analysis of polymerase chain reaction (PCR) amplified apoB VNTR locus for DNA diagnosis of heart disease. The CE separation gives an excellent resolution of two alleles differing by one or two 16 bp repeat units in the DNA size range up to 600 bp with high speed. The apoB alleles differing in length by 2 or 4 repeat units are readily distinguishable by CE in the DNA size range from 600 to 1000 bp. The plate number achieved was 1 million plates per meter. CE combining with PCR provides an excellent technique for accurate determination of the number of repeat units of apoB VNTR alleles and differentiation of heterozygous from homozygous individuals. Using the CE technique, the apoB VNTR loci from some individuals in genotyping were examined towards precise DNA diagnosis for coronary heart disease.
Influence of electrolyte composition on the effective electric field strength in capillary zone electrophoresis
Authors: Jumppanen Juho H. , Riekkola Marja‐Liisa
Published: May 2005

In capillary zone electrophoresis the electrophoretic velocity of an ion decreases as the concentration of the background electrolyte solution is increased. This is caused by changes in the electrophoretic mobility of the ion (μep) as well as by changes in the net force affecting it, namely the effective electric field strength (Eeff). The electrophoretic mobility of an ion is altered through changes in absolute viscosity of the electrolyte solution and changes in the solvated size of the ion. Eeff is altered mainly by changes in the magnitude of the charge asymmetry effect and the electrophoretic effect, both of which retard the motion of ions. In this study, the three‐marker technique was used to study the effect of background electrolyte concentration (0.02–0.08 M 3‐[cyclohexylamino]‐1‐propanesulfonic acid and counter ion (Li, Na, K, and Cs) on Eeff. It was found that the concentration of the background electrolyte markedly affects Eeff and that Eeff approaches E as the concentration of the background electrolyte approaches zero. The counter ion had a minor effect on Eeff: as the size of the hydrated radius of the counter ion increased, Eeff decreased. The three‐marker technique proved to be efficient for such determinations.
Screening of umbilical cord blood hemoglobins by isoelectric focusing in capillaries
Authors: Conti Monica , Gelfi Cecilia , Righetti Pier Giorgio
Published: May 2005

Separation and quantitation of the three main hemoglobin components of umbilical cord blood (fetal, acetylated fetal and adult hemoglobins; Hb F, Fac, A) by capillary isoelectric focusing (IEF) in a pH 6–8 gradient is reported. Even in coated capillaries (with covalently bound chains of linear acrylamido derivatives, notably N‐acryloylaminoethoxyethanol), no base line separation is obtained between Hb F and A, although this is routinely achieved in gel slab IEF. However, when the carrier ampholyte buffers were added to 3% short‐chain liquid linear polyacrylamide, base line resolution and stabilization of peak transit times were obtained. This suggests that even in the best coating procedures, patches of the inner capillary surface could still be naked, so that the static coating is complemented by a dynamic coating on the unoccupied sites. An additional improvement in separation occurs if the above mixture, comprising 5% carrier ampholytes in the pH 6–8 range and 3% soluble polyacrylamide, is made to contain 50 mM β‐alanine, a “separator” known to flatten the pH gradient around pH 7. In the normal newborns analyzed (n = 30), the following average values were obtained: Hb F, 70.1% (range 65–75%); Hb A, 20.2% (range 15–25%); and HbFac, 9.5% (range 7–11%).
Coelectrophoresis of cardiac tissue from human, dog, rat and mouse: Towards the establishment of an integrated two‐dimensional protein database
Authors: Corbett Joseph M. , Wheeler Colin H. , Dunn Michael J.
Published: May 2005

We have investigated the feasibility of identifying homologous proteins in whole tissue protein extracts of dog, mouse and rat hearts by comparison with our human heart two‐dimensional (2‐D) database. Samples of ventricular myocardial tissue from each of these species were coelectrophoresed with a human tissue sample. Gels were silver stained and patterns were analysed using PDQUEST. The number of proteins comigrating with human proteins was 301, 201 and 356 for the dog, mouse and rat, respectively. In the dog pattern, 33 of these comigrating proteins were tentatively identified from the similarity between their migration properties and those of known human proteins. Twentynine such proteins were identified in the mouse pattern while 30 comigrating rat proteins were identified. While these tentative identifications require confirmation, we feel that this technique offers a useful shortcut in the characterisation of proteins present in similar tissue samples from different species and avoids the necessity for duplicating laborious procedures, such as protein microsequencing, otherwise used in the identification of these proteins in each species.