bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of principal components analysis for mutation detection with two–dimensional electrophoresis protein separations
Authors: Taylor John , Giometti Carol S.
Published: May 2005

The application of two‐dimensional electrophoresis (2‐DE) to mutation detection requires the capability to monitor each protein in a 2‐DE pattern for significant changes in abundance indicative of a mutation event. Previously, mutation searches were done using a univariate outlier detection method in which each protein spot was considered independently in a classical outlier search. An alternative approach to analysis of 2‐DE patterns for quantitative changes is a multivariate procedure which takes advantage of the observation that protein spots in a 2‐DE pattern often represent correlated rather than independent measurements. We have compared the efficiency of univariate and multivariate procedures for mutation detection using data from the Argonne National Laboratory 2‐DE database of mouse liver proteins. Analyses involving a total of over 1500 gels were performed to compare the performance of a multivariate method based on principal components analysis (PCA) with the univariate method. Up to 279 spots from each pattern were used for PCA. First, a simulation was performed to assess the detection efficiency of PCA for single protein spots decreased in abundance by 50%. Then, the ability to detect actual mutations was tested using eight confirmed mutations. Results show that, compared to a univariate approach to analysis of data from the mouse model system, the multivariate method increases the number of protein spots on each 2‐DE pattern that can be monitored for quantitative changes indicative of mutations by compensating for variables that contribute to the background quantitative variability of protein spots.
Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti‐Borrelia burgdorferi IgG antibodies
Authors: Cruz Mabel , Sidén Åke
Published: May 2005

Anti‐Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4–10 and pH 4–7 gels. After focusing, the anti‐B.‐burgdorferi antibodies were blotted by affinity‐driven transfer to antigen‐coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG‐IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti‐B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.
Steady‐state heat transfer and thermal zone spreading in gel isoelectric focusing
Authors: Righetti Pier Giorgio , Bello Michael S.
Published: May 2005

The zone spreading caused by a transverse pH profile due to a temperature gradient through the thickness of a gel slab is estimated. The temperature difference (ΔT) between the upper and lower gel surfaces can be calculated as a function of the electric power dissipated in the gel and the gel dimensions. It is found that when ΔTis only 1 °C the zone spreading due to this thermal excursion is as high as 0.5 mm. Thus, an admissible ΔTis found to be equal to 0.2°C, since this corresponds to a thermal zone spreading of only 0.1 mm,i.e.the same order of magnitude as the spatial resolution of a laser scanner. A thermal zone spreading of 0.1 mm is compatible with a resolving power of 0.01 pH unit, the current limit of conventional isoelectric focusing in amphoteric buffers. A requirement for the thickness of a gel slab is formulated:e.g., at 40 W applied power (over a gel surface area of 25×11 cm), a thermal zone spreading limited to 0.1 mm can only be obtained with a gel thickness of approximately 170 μm.
Temperature gradient gel electrophoresis: Rapid detection of alpha‐1‐antitrypsin deficiency carriers
Authors: Hinney Anke , Luckenbach Christine , Ritter Horst
Published: May 2005

The homozygous state of the alpha‐1‐antitrypsin (αAT) deficiency variant Z is associated with severe liver damage in early childhood and progressive lung emphysema in adulthood. A single base transition (G to A in codon 342) in exon V is causing the severe disease. The Glu342 to Lys342 mutation can be detected conventionally by isoelectric focusing (IEF) or on the DNA level by the newly developed method of temperature gradient gel electrophoresis (TGGE). It is the aim of this study to describe the TGGE technique, to compare the results with conventional IEF, and to discuss its efficiency for different diagnostic applications.
A rapid and efficient method for the screening of acid phosphatase 1 in young tomato seedlings, and for the identification of root‐knot nematode species using miniaturized polyacrylamide gel electrophoresis
Authors: Cap Guillermo B. , Roberts Philip A.
Published: May 2005

A relatively rapid and highly sensitive miniaturized polyacrylamide gel electrophoresis technique is described for the analysis of certain isozymes from single cotyledons of tomato seedlings and from single females of the root‐knot nematode (Meloidogyne spp.). Homogenates from single tomato cotyledons (7, 14, 21, and 28 days old) were electrophoresed and stained for acid phosphatase 1 (Aps 1) activity. Cotyledons from plants of all the above age groups showed good Aps 1 activity. Nondestructive screening for tomato Aps 1 is therefore feasible, using very small samples, from as young as 7‐day‐old tomato seedlings. This could be of important use in expediting root‐knot nematode resistance (based on the Aps 1‐linked resistance gene Mi) screening for breeding programs, or F1 testing for seed production purposes. In addition, the mini‐polyacrylamide gel electrophoresis technique was useful for determination of the Aps 1 allelic contribution to the total enzyme activity. The system was also used to detect malate dehydrogenase and esterase isozyme activity from single adult females of the four common root‐knot nematodes, Meloidogyne arenaria, M. hapla, M. incognita, and M. javanica, with equally good results, enabling species discrimination.
Increased electrophoretic mobility of sodium sulfitetreated jack bean urease
Authors: de Llano J. Javier Martín , Gavilanes José G.
Published: May 2005

Sodium sulfite is a widely used activity‐protective agent for the storage of urease. However, this reagent produces a 10% increase in the anodic electrophoretic mobility of native urease. Changes in the hydrodynamic properties of the enzyme are not involved in that modification. The observed change is related to an increased negative charge of the protein molecule in the presence of sodium sulfite. The results are discussed in terms of sulfitolysis of the single disulfide bond in the urease monomer. It is remarkable that the modification occurs at neutral pH. Our results show that removing sodium sulfite and reversing its effect by treatment with 2‐mercaptoethanol are required prior to any study involving native urease.
Two‐dimensional electrophoretic analysis of transformation‐sensitive polypeptides during chemically, spontaneously, and oncogene‐induced transformation of rat liver epithelial cells
Authors: Wirth Peter J. , Luo Lin‐di , Fujimoto Yoshinori , Bisgaard Hanne C.
Published: May 2005

Recently, we described the establishment of a computerized database of rat liver epithelial (RLE) cellular polypeptides (Wirth et al., Electrophoresis, 1991, 12, 931–954). This database has now been expanded to include the analysis of cellular polypeptide alterations during chemically (aflatoxin B1; AFB), spontaneously, and oncogene (v‐Ha‐ras, v‐raf, and v‐myc/v‐raf)‐induced transformation of RLE cells. Two‐dimensional mapping of [35S]methionine‐labeled whole cell lysate, cell‐free in vitro translation products and [32P]orthophosphate‐labeled polypeptides revealed subsets of polypeptides specific for each transformation modality. A search of the RLE protein database indicated the specific subcellular location for the majority of these transformation‐sensitive proteins. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin‐ and intermediate filament‐related polypeptides (vimentin, ß‐tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Immunoprecipitation and Western immunoblot analysis of tropomyosin expression in four individual AFB‐, as well as four spontaneously induced, and each of the oncogene‐transformed cell lines indicated that five major tropomyosin (Tm 1–5) isoforms were variably expressed in the various cell lines, including one polypeptide tentatively identified as Tm6. Whereas alterations in tropomyosin expression appeared to be transformation‐specific, alterations in the individual intermediate filament polypeptides were related more to the differentiation state of the individual cell lines rather than to the transformation phenotype. These studies extend our earlier efforts toward the establishment of a comprehensive computerized database of RLE cellular proteins and demonstrates how such a database may serve as a useful source for studies concerning the regulation of growth and differentiation as well as transformation of RLE cells.
DNA‐protein interaction analysis using polyacrylamide gel electrophoresis and a simple and sensitive UV crosslinking procedure
Authors: Dooley Steven , Welter Cornelius , Blin Nikolaus
Published: May 2005

A simple and reproducible technique for DNA‐protein interaction analysis is described using UV crosslinking and polyacrylamide gel electrophoresis, leading to visualization of the complexes as distinct and strong signals. It avoids incorporation of bromodeoxyuridine (BrdU) into DNA and requires no special equipment. It was successfully applied to an oligonucleotide sequence from within the first intron of the mouse myb proto‐oncogene and nuclear extracts from a myb‐expressing cell line.
Inter‐α‐trypsin inhibitor polymorphism in African blacks
Authors: Vogt Ulrike , Gürtler Lutz , Cleve Hartwig
Published: May 2005

The inter‐α‐trypsin inhibitor (ITI) polymorphism was analyzed in an African Negroid population using polyacrylamide gel isoelectic focusing and subsequent immunoblotting. Gene frequencies of ITI*1, ITI*2, ITI*3 and ITI*4 were calculated to be 0.564, 0.083, 0.337 and 0.004, respectively. One unknown rare allele, ITI*6 determines further phenotypes in combination with the alleles ITI*1 and ITI*3 Gene frequency of ITI*6 was calculated to be 0.012. The common alleles are represented by ITI*1 and ITI*3. The allele distribution is therefore different from European and Asian populations.
Deletion studies to reveal the basis for size discrepancy in proliferating cell nuclear antigen
Authors: Liang Chien‐Ping , Lee Yeu‐Chin , Liu Yin‐Chang
Published: May 2005

Proliferating cell nuclear antigen (PCNA), an essential component for DNA replication in eukaryotes, is a highly conserved nonhistone nuclear protein of 261 amino acids. The molecular weight of mammalian PCNA, estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), differs notably from that predicted by the cDNA sequences, that is, 36000 in comparison with 29261 and 28748 for human and rat PCNA, respectively. To investigate if this discrepancy is due to posttranslational modifications, we studied the PCNA protein synthesized by an in vitro transcription/translation system as well as the protein overproduced in bacteria. We found that both PCNA protein samples were indistinguishable from the authentic protein from the protein mobility in SDS‐PAGE. The finding indicates that the size discrepancy is not due to the posttranslational modifications. Hence, the size discrepancy may be due to the protein sequence per se, namely a sequence‐related anomaly in SDS‐PAGE. Results from the analyses of a series of PCNA derivatives with various lengths of C‐ or N‐terminal deletion indicate that the putative sequence is in the region of residues 128–150.
Improvement of BF typing and of BF F subtyping after neuraminidase treatment in the unconverted and converted factor B
Authors: Siemens Ingbert , Geserick Gunther , Mauff Gottfried , Correns Andreas , Schröder Helga
Published: May 2005

When neuraminidase‐treated sera are analyzed by agarose gel isoelectric focusing, the factor B (BF) banding pattern is reduced to predominantly one major band without cathodically positioned bands. This not only makes unequivocal typing of BF allotypes possible but also the reliable distinction of all BF F subtype phenotypes with delimitation of “BF F subtype variants”. With this new method, serum aging affects the BF determination to a lesser extent than when applying methods that separate native sera. We show that sialylation is not responsible for the BF F subtype polymorphism. All of the investigated BF allotype bands, including those characteristic of the subtypes, show functional hemolytic activity. The banding pattern after removal of neuraminic acid residues ranges from pH 6.8 to 7.3 for factor B, from pH 5.3 to 5.9 for the Ba fragment, and from pH 8.2 to 8.7 for the Bb fragment. The protein structure of factor B is also discussed. Eliminating the superimposition of bands in different BF allotypes, as demonstrated by these methods, proved to be necessary for the detection of hypomorphic BF gene products (BF QL), which are expressed by assumed BF*Q0 alleles in heterozygous genotypes. This allows investigation of BF*Q0 alleles on a protein level, which complements molecular genetic approaches.
v‐erbB oncogene expression accounts for most variations in protein synthesis after avian erythroblastosis virus infection of chicken embryo fibroblasts: A two‐dimensional electrophoresis study
Authors: Desbois Christelle , Gandrillon Olivier , Samarut Jacques , Madjar Jean‐Jacques
Published: May 2005

The effect of the v‐erbA and/or v‐erbB oncogenes on cellular gene expression was investigated after separation by two‐dimensional polyacrylamide gel electrophoresis of [35S]methionine‐labelled proteins from chicken embryo fibroblasts (CEF), infected by either the avian erythroblastosis virus (AEV) carrying both oncogenes, or by viruses carrying only one of them. We observed significant changes in the synthesis of 34 proteins in AEV‐transformed CEF as compared with control cells. The synthesis of 24 of them was increased while the synthesis of the other 10 proteins was decreased. The expression of v‐erbB alone is necessary and sufficient to induce changes in the synthesis of 27 proteins while the 7 remaining modifications are observed only in cells expressing v‐erbB together with v‐erbA. Moreover, the deregulation of protein synthesis by v‐erbB‐expressing viruses was correlated with the morphological transformation state of cells.
A new concept for sequencing DNA by capillary electrophoresis
Authors: Noolandi Jaan
Published: May 2005

It is proposed that the scaling symmetry of constant charge density with increasing molecular weight, which prevents the separation by electrophoresis of DNA molecules in solution (with respect to molecular weight) be broken by the attachment of a perturbing entity (protein, virus or charged sphere) to one end of the molecule. An application of this idea to a concept for sequencing DNA by capillary electrophoresis is discussed, and the possibility of using the reattachment of the RecA protein to separate large segments of DNA in solution by electrophoresis following sequence‐specific cleavage is mentioned.
Separation of proteins by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of soluble, aqueous polymers: Ficoll and polyvinylpyrrolidone
Authors: Gersten Douglas M. , Bijwaard Karen E.
Published: May 2005

In previous studies we have demonstrated that water soluble polymers of dextran and methylcellulose, when incorporated into sodium dodecyl sulfate (SDS)‐polyacrylamide gels, can be used to sharpen the protein bands. We have extended these studies to enhance the separations in the mid‐molecular weight range. Enhanced separation of protein molecular weight markers between 30000–67000 was best achieved in SDS‐gels containing 2.5% polyvinylpyrrolidone (average molecular weight 10000).
Classification of crossed immunoelectrophoretic patterns using digital image processing and artificial neural networks
Authors: Søndergaard Ib , Krath Britta Naimi , Hagerup Martin
Published: May 2005

A method is presented which makes it possible to present crossed immunoelectrophoretic patterns to an artificial neural network. The electrophoretic patterns are presented for the artificial neural network as three‐dimensional vectors and it is shown that it is possible with this representation to train the network to learn the patterns and classify them. It was found that the ability to generalize was substantially increased by the addition of noise to the input patterns during training. Furthermore, the addition of noise decreased the number of presentations needed to reach the predetermined error level. The trained neural network was able to classify all distorted patterns correctly within an error range of 1%.
Sex identification by polymerase chain reaction of α‐satellite in aged tissue samples
Authors: Fattorini Paolo , Cacció Simone , Gustincih Stefano , Florian Fiorella , Altamura Bruno M. , Graziosi Giorgio
Published: May 2005

In vitroamplification of the alphoid repeated sequences clustered in the centromeric regions of the human × and Y chromosomes was carried out. A modification of the amplification conditions reported by Witt and Erickson [1] gave clear amplification bands from 10 pg of genomic DNA template. Results were obtained from the following aged tissue samples: 1 μL three‐year‐old blood stain, one‐year‐old single hair roots, six‐month‐old saliva stains and twelve‐year‐old bone fragments. Good results were obtained even when the template was artificially fragmented into fragments of less than 1 kbp.
Gel electrophoresis of giant proteins: Solubilization and silver‐staining of titin and nebulin from single muscle fiber segments
Authors: Granzier Henk L. M. , Wang Kuan
Published: May 2005

Giant proteins in the megadalton range (> 0.5 MDa) appear to play important structural and functional roles in striated muscle. Titin (∼ 3 MDa) is involved in the generation of resting tension and the assembly and stability of the sarcomere in skeletal and cardiac muscle tissues, while nebulin (∼ 0.7 MDa) is thought to regulate thin filament length in skeletal muscle. Sodium dodecyl sulfate (SDS)‐gel electrophoresis is an important tool in revealing the size, quantity and integrity of these giant proteins in muscle tissues. We report here a method for solubilizing, detecting and quantifying titin and nebulin from short segments of single fibers of the rabbit psoas muscle. Muscle proteins ranging from 15 kDa to 3MDa were resolved on 3.3–12% gradient polyacrylamide gels that were silver‐stained and quantitated by densitometry. Presoaking fiber segments in a low ionic strength pH 8.4 buffer enhances the amount of solubilized titin and nebulin. Solubilizing the presoaked fiber segments with SDS at 60°C for 60 s maximizes the amount of intact titin; solubilizing at higher temperatures causes extensive degradation of titin. Detection sensitivity is sufficient to study titin and nebulin in fiber segments as short as 120 μm.
Extracellular protein secretion of cultured normal and Pagetic osteoblasts
Authors: Hankey David P. , Hughes Anne E. , Mollan Raymond A. B. , Nicholas Richard M.
Published: May 2005

Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) has been used to map the extracellular secretory activity of normal osteoblasts. The proteins osteonectin, bone sialoprotein, and both the C‐telopeptide of collagen I together with collagen I have now been positionally identified. In addition the secretory differences which exist between normal and Pagetic osteoblasts have been mapped with the Pagetic osteoblasts shown to consistently secrete an altered 30 kDa C‐telopeptide of collagen type I. The use of the diphosphonate Pamidronate in the treatment of Paget's disease of bone has beneficial effects with suppression of the bone isoenzyme marker alkaline phosphatase. It has been reported that diphosphonates directly inhibit human osteoblast secretory function as well as osteoclast metabolism. The effects of Pamidronate on the secretory activity of normal and Pagetic osteoblast cultures was also investigated. The extracellular protein secretion of normal and Pagetic osteoblasts was not affected by Pamidronate treatment as assessed by 2‐D PAGE. This technique allows a comprehensive multiparameter assessment of extracellular secretory activity in normal and diseased states. The findings show that the Pagetic osteoblasts cultured in vitro are functionally abnormal and they support the hypothesis that the underlying problem in Paget's disease is characterised by disorder of osteoblast and osteoclast interactions.
Rapid detection of mitochondrial DNA polymorphism by photoactivatable biotin
Authors: Tsuchida Shuichi , Ikemoto Shigenori
Published: May 2005

A new method for the detection of mitochondrial (mt)DNA polymorphism by agarose gel electrophoresis is presented. In this method, after mtDNA digestions with endonucleases, the mtDNA fragments are labeled directly with photoactivatable biotin in the digestion buffer, separated by agarose gel electrophoresis, transferred to a nylon membrane and detected enzymatically using streptavidinalkaline phosphatase, Nitroblue tetrazolium (NBT) and 5‐bromo‐4‐chloro‐3‐indolylphosphate (BCIP). This method is useful for the detection of mtDNA digestion patterns by agarose gel electrophoresis, especially when enough purified mtDNA cannot be obtained and the radioisotope cannot be handled because of several restrictions.
Detection of DNA damage‐recognition proteins using the band‐shift assay and Southwestern hybridization
Authors: Protić Miroslava , Levine Arthur S.
Published: May 2005

We describe electrophoresis and biochemical conditions that allow detection of damaged DNA‐binding proteins in cell extracts. In addition, we present an overview of the damage‐recognition DNA‐binding proteins from eukaryotic cells and discuss their hypothetical role in DNA repair.
Electrophoretic analysis of protein‐induced DNA bending and twist changes
Authors: Niederweis Michael , Hillen Wolfgang
Published: May 2005

DNA fragments with a varied phasing of two intrinsic bends at their ends and a tet operator in‐between were constructed to determine Tet repressor‐induced twist changes in the operator DNA. These distance variants show a sinusoidal dependence of their electrophoretic mobilities on the phasing of their bends in polyacrylamide gels. Complex formation with Tet repressor leads to a displacement of the first minimum, indicating an unwinding of the tet operator DNA. Model calculations were performed to assess the contribution of Tet repressor‐induced DNA bending to this result. They revealed that the amplitude of the electrophoretic mobilities of the distance variants may be used as a parameter to separate the effects of Tet repressor‐induced twist change and bending. The systematic analysis presented here may help to improve the quantitative interpretation of gel shifts and promote the use of this highly sensitive method to gain structural information about protein‐DNA complexes.
Automated Ferguson analysis of glycoproteins by capillary electrophoresis using a replaceable sieving matrix
Authors: Werner William E. , Demorest David M. , Wiktorowicz John E.
Published: May 2005

Obtaining accurate molecular weight estimates for glycoproteins by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) has been difficult due to the lack of SDS binding by the carbohydrate moieties of the proteins. This leads to lower charge‐to‐mass ratios for SDS‐glycoprotein complexes, resulting in over‐estimation of molecular weights by SDS‐PAGE. In order to minimize these inaccuracies for proteins with abnormal charge‐to‐mass ratios, a Ferguson plot may be employed. This application requires the determination of relative mobilities for standard proteins in addition to unknowns at several different gel concentrations. Historically, this technique has not been popular because it requires time‐consuming preparation of gels with varying matrix concentrations, electrophoresis, and staining/destaining of gels. In this paper a procedure is demonstrated which automatically generates all of the data required for a Ferguson plot using a replaceable sieving matrix (thereby eliminating gel polymerization) in a capillary format. In addition, this technique possesses the advantages inherent to capillary electrophoresis, namely, very fast separation times, and on‐line monitoring which allows quantitation and precludes post‐separation staining and destaining of gels.
Identification of α‐fetoprotein‐P4 sugar chain as α 2→6 monosialylated biantennary complex‐type oligosaccharides with an exposed galactose on the mannose α 1→6 arm by two‐dimensional extended agarose gel‐lectin affinity electrophoresis
Authors: Taketa Kazuhisa , Fujii Yasuto , Aoi Toshie , Taga Hiroko , Nishi Shinzo
Published: May 2005

Erythroagglutinating phytohemagglutinin (E‐PHA)‐reactive α‐fetoprotein (AFP)‐P4 and E‐PHA weakly‐reactive AFP‐P3 of sera from cord blood at term and from patients with hepatocellular carcinomas showed a mobility of monosialo‐AFP in two‐dimensional extended agarose gel‐E‐PHA affinity electrophoresis. Asialo‐AFP had as high an affinity for E‐PHA as that of monosialo‐AFP‐P4, while disialo‐AFP, AFP‐P2, revealed a negligible affinity for E‐PHA. For Allomyrina dichotoma lectin (allo A), asialo‐AFP had no affinity with a mobility of AFP‐A1s, monosialo‐AFP‐P4 had the highest affinity with a mobility of AFP‐A3, and monosialo‐AFP‐P3 had an intermediate affinity with a mobility of AFP‐A2s. The affinity of AFP‐P4 for E‐PHA was reduced by galactosidase digestion, indicating the presence of galactose residues, one sialylated and the other exposed. From the known high affinity of E‐PHA for biantennary complex‐type oligosaccharides with a terminal β 1→4 galactose of the Man α 1→6 antenna and from that of allo A for an α 2→6 sialylated β 1→4 galactose of the Man α1→3 antenna, together with the nonbisected biantennary structure of AFP‐P4 as deduced from its high affinity for concanavalin A, the sugar chain structure of AFP‐P4, which increases frequently in hepatocellular carcinomas, was identified as α 2→6 monosialylated nonbisected biantennary complex‐type oligosaccharides with an exposed β 1→4 galactose of the Man α 1→6 arm and with or without fucosylated core N‐acetylglucosamine, and AFP‐P3 as similar oligosaccharides with an exposed β 1→4 galactose of the Man α 2→3 arm.
Electrophoretic characterization of wheat grain allergens from different cultivars involved in bakers' asthma
Authors: Weiss Walter , Vogelmeier Claus , Görg Angelika
Published: May 2005

In order to identify and characterize the wheat grain allergens involved in bakers' asthma, proteins were sequentially extracted from whole‐meal flour. The polypeptide composition of the individual solubility fractions (albumin/globulin, gliadin and glutenin) was analyzed by sodium dodecyl sulfate‐gel electrophoresis (SDS‐PAGE), and high‐resolution two‐dimensional gel electrophoresis with immobilized pH gradient 4–9 in the first dimension (IPG‐Dalt). The resolved polypeptides were transferred onto an immobilizing polyvinylidene difluoride membrane and incubated with a pooled serum from four asthmatic bakers. Bound IgE was demonstrated by autoradiography using125I‐labeled anti‐human IgE. Our study demonstrated that the serum of the bakers allergic to flour contained IgE antibodies which bound to numerous polypeptides of all three solubility fractions. The highest percentage of IgE binding was observed with certain albumin and/or globulin polypeptides, whereas the gliadins and glutenins exhibited considerably less allergenicity. SDS‐PAGE revealed that the protein which bound the highest percentage of IgE from the sera of the allergic bakers is a 27 kDa albumin. More detailed investigations using IPG‐Dalt demonstrated that this allergen is not a single polypeptide but consists of several polypeptide spots that differ in their isoelectric points. Quantitative studies using computer‐assisted laser densitometry revealed that the amount of patients' IgE bound by these particular polypeptides differed considerably between the seven wheat cultivars examined, ranging from 13% to 53% of the total radioactive uptake.
Semi‐dry PhastTransfer detection of biotinylated cell surface molecules
Authors: Abdi Kaveh , Li Xiaopu , Mentzer Steven J.
Published: May 2005

The PhastTransfer system is a semi‐dry electrophoretic unit designed to optimize the transfer of small amounts of protein. Because of its efficiency, we adapted the PhastTransfer system for the detection of labeled membrane molecules. Biotin was used as the membrane molecule label because it permitted the long‐term storage of labeled lysates as well as the flexibility of derivatizing several different functional groups. After immunoaffinity separation using magnetic microspheres, the protein was electrophoretically separated with the PhastSystem and transferred with the PhastTransfer unit. Using an avidin‐linked enzyme amplification system, less than 10 ng of loaded protein could be detected on the transfer membrane. Based on these findings, the PhastTransfer system is a fast, reproducible, and convenient method for the transfer of small quantities of labeled cell surface protein.
Electroblotting proteolytic products from native gel for direct N‐terminal sequence analysis: An approach for studying protein‐protein interaction
Authors: Wang Feng , Su Chang , Hollfelder Kurt , Waddington Doreen , Pan Yu‐Ching E.
Published: May 2005

Proteins which are electroblotted from native gels onto polyvinylidene difluoride (PVDF) membranes are suitable for detailed structural analysis. This method, in conjunction with limited proteolysis and N‐terminal sequencing, has been used to study the molecular interactions between native protein molecules. The interaction between recombinant interleukin‐2 (rIL‐2) and its receptor (rIL‐2Rα) was examined as a model system. The working strategy consists of (i) proteolysis of rIL‐2Rα and rIL‐2Rα/rIL‐2 complex, (ii) separation of the major proteolytic products by native polyacrylamide gel electrophoresis followed by electroblotting onto PVDF membrane, and (iii) sequence analysis of the blotted protein bands for the identification of peptide regions sensitive to proteolysis. Results have indicated that the exon 3 encoded region in rIL‐2Rα is sensitive to proteolysis regardless whether it is complexed with rIL‐2 or not. This suggests that no major conformational changes occur in rIL‐2Rα during interaction with rIL‐2. This electroblotting approach is, therefore, useful for studying protein‐protein interaction in solution.
Skimmed‐milk blocking improves silver post‐intensification of peroxidase‐diaminobenzidine staining on nitrocellulose membrane in immunoblotting
Authors: Ludány Andrea , Gallyas Ferenc , Gaszner Balázs , Andrásfalvy Bertalan , Szücs György , Kellermayer Miklós
Published: May 2005

A silver‐staining procedure for enhancing the sensitivity of protein detection on nitrocellulose membranes in immunoblotting is described. After completing any peroxidase‐Ni‐diaminobenzidine immunostaining, nitrocellulose sheets are placed in a physical developer, containing sodium tungstate and ascorbic acid, until the desired grade of silver‐intensification has been reached. In this way a 16‐ to 64‐fold amplification of intensity of the initial immunostaining can be achieved. False positive silver staining of protein bands and of background are suppressed by replacing bovine serum albumin, the blocking agent most frequently used in immunoblotting, with skimmed milk.
Antibodies bound to nitrocellulose in acidic buffers retain biological activity
Authors: Hoffman Wayne L. , Jump Adrien A. , Ruggles Ann O. , Kelly Patrick J.
Published: May 2005

This report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in both acidic and neutral buffers. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on the pH of the buffer used to bind the proteins to the membrane. Most proteins that bound to nitrocellulose in acidic buffers were relatively resistant to milk‐stripping compared to proteins bound in pH 7 buffer. Acid‐binding of proteins to nitrocellulose also decreased the amount of protein that was stripped from the nitrocellulose membrane when Tween 20 was included in the washing buffer. After correcting for the amount of antibody remaining on the membrane after the milk block, it was found that acid‐bound antibodies were unchanged in biological activity when compared with the same antibodies bound at neutral pH. These results suggest that acid‐binding of proteins could increase the sensitivity of nitrocellulose membrane assays that use milk and/or Tween 20.
Intra‐species cross‐reactivity of house dust mite allergens separated by protein blotting and detected by selective elution of mite components and IgE antibodies
Authors: O'Neill Geraldine M. , Baldo Brian A.
Published: May 2005

A complex and varied antibody response to allergens, in particular Dermatophagoides pteronyssinus, is observed in the serum of allergic patients. To examine the role of cross‐reacting antigenic determinants in allergy, a strategy has been devised for isolating antibody populations that bind to individual polypeptides of an allergen source separated by sodium dodecyl sulfate‐polyacrylamide electrophoresis and electroblotted to nitrocellulose. Polypeptides are identified on the blots, desorbed and coupled to CNBr‐activated paper discs. The coupled discs are then used as immunoadsorbent surfaces for the binding of antibodies from allergic sera. Antibodies eluted from the polypeptide immunoadsorbents are then used to reprobe protein blots of the allergen. Results are presented which demonstrate strong cross‐reaction between two major mite allergens, Der p I and Der p III, and some weaker intra‐species cross‐reactivities.
Immune response to a hapten of fluorescein isothiocyanate in a single mouse analyzed by two‐dimensional affinity electrophoresis
Authors: Nakamura Kazuyuki , Mimura Yusuke , Takeo Kazusuke
Published: May 2005

Immune response to a hapten of fluorescein isothiocyanate (FITC) in a single BALB/c strain mouse was analyzed by two‐dimensional affinity electrophoresis (2D‐AEP). Anti‐FITC antibodies were induced by immunization with FITC‐conjugated bovine serum albumin. The antibodies were separated into a large number of spots of IgG due to differences in their isoelectric points(pI) and binding affinities to the FITC ligand. These spots consisted of IgG families which were composed of several spots having an identical affinity to the ligand but a different pI. The spots were not clearly detected in the antiserum taken on day 7 after the primary immunization, but on day 21 the spots of IgG were clearly detected, with a high diversity and specificity for the ligand. The size and number of IgG spots were markedly increased by the secondary immunization; however, the third immunization did not increase the size and number of IgG spots. The IgG spots of each family were specifically stained with an antimouse IgG subclass antibody. Furthermore, a monoclonal antibody (FL‐D6) was separated by 2D‐AEP into a single family which consisted of seven IgG1 spots having an identical affinity to FITC but different pIs. Therefore, each of the IgG families of anti‐FITC antibodies in the antiserum can be generated by a single clone of anti‐FITC antibody‐producing cells. The substitution of dextran T 2000 or lipopolysaccharide for bovine serum albumin as a carrier for FITC induced much smaller amounts of anti‐FITC antibodies with a low diversity but high specificity to FITC.
Humoral immune response against Helicobacter pylori as determined by immunoblot
Authors: Faulde Michael , Cremer Josef , Zöller Lothar
Published: May 2005

An immunoblot method has been evaluated to diagnose Helicobacter pylori infection serologically by comparing 69 serum specimens from patients with a positive Gram stain and/or culture result and a positive urease test on biopsy material, as well as 51 serum specimens from patients with at least 4 negative urease tests, and negative microscopy and culture results. Sensitivity and specificity was found to be 100%. Recognition of the cross‐reacting flagellin (66 kDa), flagellar sheath protein (51 kDa), and a 14 kDa protein are not a criterion for a current H. pylori infection. On the other hand, any combination of at least two of the 180, 120, 90, 75, 67, 29.5 and 19 kDa bands were diagnostic of infection. Three H. pylori strains, which were compared with both gel electrophoretic analyses and immunoblot reactivity, exhibited in part strong qualitative and quantitative differences that particularly affect the 120 kDa pathogenic factor, the large urease subunit and other proteins especially in the molecular mass range from 50 to 67 kDa. IgG immunoblot patterns showed that the choice of H. pylori strain, as well as a reproducible and standardizable antigen preparation, is of great importance for the reliability of serodiagnostic tests. The immunoblot method was found to be a valuable tool for the semiquantitative confirmation of results achieved with other serological methods as well as optimization and quality control of the antigens used for serodiagnostic purposes.
On the limits of near‐equilibrium DNA gel electrophoretic sequencing
Authors: Slater Gary W. , Mayer Pascal , Drouin Guy
Published: May 2005

High field intensities deeply affect the migration mechanisms of DNA in gels. The corresponding large electric forces lead to far‐from‐equilibrium transport properties such as the limiting mobility, increased diffusion and strong pulsed field responses. We present a theoretical study of the conditions required to retain a near‐equilibrium reptation transport behaviour. We then examine the possibility of increasing both the field intensity and the gel concentration to sequence large single‐stranded DNA molecules rapidly and efficiently. Finally, we discuss possible experimental approaches based on our theoretical predictions.
Simple repetitive (GAA)n loci in the human genome
Authors: Siedlaczek Ina , Epplen Cornelia , Rieß Olaf , Epplen Jörg T.
Published: May 2005

In order to investigate the organization (and inheritance) of simple tendem (GAA)n repeats in the human genome, different restriction enzymes were employed for DNA digestion followed by separation of the resulting fregments by agarose gel electrophoresis. Frequently cutting enzymes (4 bp recognition sites) revealed highly complex multilocus banding patterns after conventional horizontal submarine gels. Fragments larger than 25 kb, resulting from digestions with rarely cutting enzymes (6 bp recognition sites), were separated by pulsed‐field gel electrophoresis (PFGE). Hybridizations were carried out directly in the gel matrix. Nearly all of the enzymes produce at least one predominant signal band after hybridization with (GAA)6. The frequency of (GAA)n stretches was estimated on human chromosome 4 by probing a respective cosmid library. In comparison with other simple di‐, tri‐ and tetranucleotide repeats, (GAA)n stretches appear underrepresented. Hybridization of a fetal human brain cDNA library indicated very few expressed (GAA)n repeats. These data are discussed with particular reference to genomic organization and other simple repetitive trinucleotide stretches.
The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences
Authors: Bjellqvist Bengt , Hughes Graham J. , Pasquali Christian , Paquet Nicole , Ravier Florence , Sanchez Jean‐Charles , Frutiger Séverine , Hochstrasser Denis
Published: May 2005

The focusing positions in narrow range immobilized pH gradients of 29 polypeptides of known amino acid sequence were determined under denaturing conditions. The isoelectric points of the proteins calculated from their amino acid sequences matched with good accuracy the experimentally determined pI values. We show the advantages of being able to predict the position of a protein of known structure within a two‐dimensional gel.
Optimizing a method of protein extraction for two–dimensional electrophoretic separation of proteins from planarians (Platyhelminthes, Turbellaria)
Authors: Collet Joan , Baguñà Jaume
Published: May 2005

Different procedures for microscale extraction of proteins from small amounts of tissue of planarians (Platyhelminthes, Turbellaria) to be analyzed by two‐dimensional gel electrophoresis (2‐D PAGE) are compared. Three extraction methods were assessed: (i) extraction of soluble proteins with nondenaturing Tris buffers, (ii) extraction with Tris buffer containing the anionic detergent sodium dodecyl sulfate (SDS), and (iii) denaturing extraction under reducing conditions in the presence of urea and Nonidet P‐40 (NP‐40) with or without SDS. Buffers combining minute concentrations of SDS (0.01%), denaturing concentrations of urea (8M) and alkaline pH solubilized the greatest number of proteins without detectable proteolysis. Neither the presence of protease inhibitors nor higher concentrations of SDS improved protein extraction. We have applied this method to planarians to detect proteins specific to the pharynx. The resulting two‐dimensional pattern shows a larger number of specific spots than in previous extraction methods.
Electrophoretic analysis of plant cysteine and serine proteinases using gelatin‐containing polyacrylamide gels and class‐specific proteinase inhibitors
Authors: Michaud Dominique , Faye Loïc , Yelle Serge
Published: May 2005

Inclusion of gelatin in polyacrylamide gels provides a sensitive way of detecting multiple proteolytic activities in crude extracts from any source. The present study describes a method allowing discrimination between cysteine and serine proteinases in plant extracts, using gelatin‐containing gels in combination with class‐specific proteinase inhibitors. Preincubation of extracts with 4 mM phenylmethylsulfonyl fluoride, a serine proteinase inhibitor, or with 25 μM L‐trans‐epoxysuccinyl‐L‐leucylamido(4‐guanidino) butane, a cysteine proteinase inhibitor, allowed the identification of enzymes from both classes in extracts of tomato fruit and papaya latex. The efficiency of the two low molecular weight inhibitors used was very high, and the irreversibility of the inhibiting effect was maintained during electrophoresis conducted in the presence of sodium dodecyl sulfate. The analytic procedure described here, with a detection threshold of less than 100 pg enzyme, is the first that allows quick and accurate discrimination of plant cysteine and serine proteinases separated in electrophoretic gels. This simple and rapid technique could be of interest for studying the evolution of class‐specific proteinases in plant extracts during various developmental, physiological, and pathogenic processes. It is also potentially applicable to the majority of eucaryotic and procaryotic systems.
Precise quantitation of chloramphenicol acetyl transferase reporter mRNA by competitive polymerase chain reaction
Authors: Greco Anna , Massé Thierry , Madjar Jean‐Jacques
Published: May 2005

A strategy, based on competitive polymerase chain reaction (PCR) after reverse transcription, was developed to quantitate mRNA of the E. coli chloramphenicol acetyl transferase (CAT) gene. Precise quantitation of this rare mRNA is achieved by co‐amplification of a constant amount of the complementary DNA (cDNA) with successive dilutions of a competitive DNA template of known concentration and with the use of the same primers. The unique EcoRI site located in the CAT coding sequence has been abolished in the competitive DNA. The two oligonucleotide primers used are both located in the CAT coding sequence at equal distance from the EcoRI site. After amplification, the PCR products from the cDNA to be quantified and from the competitor DNA are discriminated by EcoRI digestion, followed by separation of the resulting fragments by agarose gel electrophoresis. DNA amplified from the target cDNA is the only DNA digested by EcoRI into two fragments of identical size co‐migrating in an agarose gel. After ethidium bromide staining, comparison of the intensity of fluorescence of the resulting bands in a competition series permits us to estimate precisely the amount of CAT cDNA and therefore of the mRNA to be quantified.
Characterization of E‐PHA‐reactive α‐fetoprotein isoforms by two‐dimensional lectin affinity electrophoresis
Authors: Taketa Kazuhisa , Fujii Yasuto , Taga Hiroko
Published: May 2005

Erythroagglutinating phytohemagglutinin (E‐PHA)‐dependent isoforms of human α‐fetoprotein (AFP) from cord blood were analyzed for their carbohydrate structures by two‐dimensional electrophoresis with E‐PHA combined with extended agarose gel electrophoresis or with affinity electrophoresis with concanavalin A or Allomyrina dichtoma lectin. By means of neuraminidase and/or β‐galactosidase treatment, AFP‐P2 was identified as α2→6 disialo‐AFP, AFP‐P3 as having biantennary structures with α2→6 monosialylated galactose of the Mannose (Man) α1→6 arm, AFP‐P4 as having α2→6 monosialylated galactose of the Man α1→3 arm, and AFP‐P5 as disialo‐AFP with α2→3 sialylated galactose of the Man α1→6 antenna and with α2→6 sialylated galactose of the other antenna. Desialylated AFP with the terminal galactose of the Man α1→6 antenna with or without the galactose of the other arm also had a migration of AFP‐P4, and other hydrolytic intermediates without the terminal galactose of the Man α1→6 arm with and without the galactose of the other antenna had mobilities of AFP‐P3s and AFP‐P3, respectively. Thus, the present system of two‐dimensional lectin affinity electrophoreses would provide a model for the determination of the sugar chain structure of glycoproteins.
Affinity maturation of anti‐hapten antibodies in a single mouse analyzed by two‐dimensional affinity electrophoresis
Authors: Nakamura Kazuyuki , Mimura Yusuke , Tanaka Tatehiko , Fujikura Yoshihisa , Takeo Kazusuke
Published: May 2005

Affinity maturation of anti‐hapten antibodies in an inbred mouse (BALB/c) was analyzed by two‐dimensional affinity electrophoresis (2D‐AEP) in which the antibodies specific to a hapten of dinitrophenyl (DNP) group were separated into a large number of IgG families according to the differences in their isoelectric points (pI) and in their affinities for the ligand of DNP immobilized in the gel matrices. Each of the IgG families consisted of several spots showing an identical affinity for the ligand but different pI, and the spots were specifically stained by immunostaining with an anti‐murine IgG subclass antibody. These results lead us to the conclusion that each of the IgG families is derived from a single clone of antibody‐producing cells specific to DNP. The mass and affinity of the IgG families varied in the course of immunization with DNP‐conjugated chicken serum albumin (DNP‐CSA). The first injection of DNP‐CSA induced a small amount of IgG families showing a wide variety in their affinity for DNP. The second injection induced a large amount of IgG families, especially of families having a medium affinity for DNP. Additional injections, however, did not change the mass or affinity of the IgG families significantly. This suggests that an antigen‐induced somatic mutation of the immunoglobulin gene does not occur frequently to mature the affinity of antibodies by the additional injections after the second injection. 2D‐AEP enables us to analyze the affinity maturation of antibodies in vivo in a single mouse and to document the subclass switch of the antibody in the course of immunization.