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  • Two-dimensional polyacrylamide gel electrophoresis  (12)
  • Wiley-Blackwell  (12)
  • German Medical Science GMS Publishing House; Düsseldorf
  • 1
    ISSN: 0173-0835
    Keywords: Galectins ; Hematopoietic cells ; Two-dimensional polyacrylamide gel electrophoresis ; Immunoblotting ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Vertebrate soluble β-galactoside-binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross-reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), even when followed by immunoblottin. However, their different isoelectric points allow their identification using isoelectric focusing and two-dimensional (2-D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2-D immunoblotting using anti-galectin 1 (Gal-1) and anti carbohydrate-binding protein 15 (CBP15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG-Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside-binding proteins even at basic p/s.
    Additional Material: 8 Ill.
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  • 2
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Protein sequencing ; Allelic variations ; Genome mapping ; Maize ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Mapping cDNA probes in order to construct genetic linkage maps is becoming a widespred strategy for genome analysis and gene isolation, particularly in cultivated plant species. Nevertheless, almost all cDNAs reveal two or more unlinked loci, making it difficult to identifiy the gene(s) actually expressed. In a highly polymorphic species, such as maize, two-dimnsional polyacrylamide gel electrophoresis (2-D PAGE) of proteins may circumvent this limitation. With the analysis of various segregating populations, we previously showed that the apparent position shifts of proteins on the 2-D gels are monogenic and codominant. In this paper we compared allelic proteins on the basis of their high performance liquid chromatography (HPLC) profile and partial amino acid sequences. In a sample of 20 position shifts, the allelic proteins appeared to be similar in all but one case, strongly suggesting that polymorphism of structural genes is involved. Thus 2-D PAGE could prove to be a useful tool for genome mapping: when a cDNA probe detects several loci, a position shift of the encoded protein will allow the identification of the gene translated in the organ considered.
    Additional Material: 5 Ill.
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  • 3
    ISSN: 0173-0835
    Keywords: Triticum ; Secale ; Hordeum ; Two-dimensional polyacrylamide gel electrophoresis ; Phylogenetic relationships ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: 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.
    Additional Material: 3 Ill.
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  • 4
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Phagocytosis ; Lysosome ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Phagosomes are the organelles formed de novo in a variety of cells by the internalization of large particulate materials, including a wide range of pathogenic microorganisms. We present here a systematic approach that can be used to study the polypeptide composition of phagosomes/phagolysosomes and to yield analytical information on the characteristics of their proteins. A density shift approach was used to isolate pure preparations of phagosomes filled with low density latex beads from mouse J774 and human U937 macrophages. High resolution two-dimensional (2-D) gel electrophoresis was performed to generate a map of the overall [35S]methionine-labeled protein profile of the isolated phagosomes. The resulting map showed the minimal presence of over 200 polypeptides, indicating the complexity of this organelle. Comigration experiments showed that several phagosome polypeptides, among them several known proteins, are shared by the two species. Extraction with Triton X-114 and sodium carbonate was performed to distinguish between membrane and soluble proteins, and sensitivity to a panel of proteases was measured to identify proteins exposed on the cytoplasmic face of the phagosome membrane. The general value of the 2-D gel approach in the mapping of organelle proteins is discussed.
    Additional Material: 6 Ill.
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  • 5
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Protein identification ; Amino acid composition ; Maize ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The possibility of using experimentally determined amino acid composition to assess relatedness between 75 proteins separated by two-dimensional electrophoresis (2-DE) and to identify them was tested on maize. Two independent parameters, the relative Euclidean distance and the correlation coefficient between the amino acid compositions, were evaluated and used. Previous sequence information for 31 out of the 75 proteins made it possible to evaluate the method for the detection of isoforms and for identification. However, the extension of the interrogation beyond maize to all plant sequences raised the problem of false positives that could nevertheless be limited by replications and by using additional information. The efficiency of the method to assess relatedness betwen proteins should make amino acid composition analysis a valuable tool in large protein characterization programs based on 2-DE, by facilitating the transfer of information from one well-documented organ/tissue or genotype to another.
    Additional Material: 4 Ill.
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  • 6
    ISSN: 0173-0835
    Keywords: Durum wheat ; Two-dimensional polyacrylamide gel electrophoresis ; Varietal identification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Four closely related curum wheat varieties were compared by computer-assisted analysis of two-dimensional electrophoretic maps of leaf proteins. A low inter-varietal polymorphism was revealed and seven reliable qualitatively varying proteins allowed rapid visual identification of genotypes. For numerous spots, presence/absence or quantitative variations were greatly affected by a batch effect. Several criteria that should be used to discard unreliable spots or gels a priori were reviewed. Nevertheless, it was shown that, provided that the experimental design allows the integration of the batch effect, screening for discriminant markers as well as computing distances based on protein quantity variations are possible and allow variety identification. Euclidean and Mahalanobis distances allowed variety discrimination and single gel classification with a minimum risk of error, not only by taking into account the quantitative variations in discriminant proteins selected by analysis of variance, but also by taking into account all reproducible spots. The possible applications of two-dimensional electrophoresis in variety identification are discussed.
    Additional Material: 3 Ill.
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  • 7
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Brucella ; Immunoblotting ; Microsequencing ; Immunogenic proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In a previous report, proteins from Brucella melitensis were characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and N-terminal microsequencing. In the present report, we have extended this study to the second etiologic agent in ovine brucellosis, B. ovis, responsible for ram epididymitis and infertility. The combination of 2-D gel electrophoresis and protein microsequencing facilitated the location and identification of the major proteins of B. ovis on the 2-D pattern. These proteins comprised cytoplasmic, periplasmic and some membrane proteins except the major outer membrane proteins. By comparing 2-D gel profiles of B. ovis with that of B. melitensis described previously, a few proteins with different expression levels were readily identified. Serum from a ram naturally infected with B. ovis was used in immunoblotting studies to identify immunogenic proteins recognized during the course of infection. This serum showed antibody reactivity against approximately 82 protein spots. Twenty-one of these proteins were identified either by use of monoclonal antibodies or by N-terminal microsequencing. Several proteins previously described in earlier Brucella works were identified: the 89 kDa outer membrane protein, DnaK, GroEL, BP26, and Cu-Zn superoxide dismutase. Eight proteins had amino acid sequences homologous to those of various proteins from other bacteria found in protein databases: NikA, dihydrolipoamide succinyltransferase, a hypothetical 31 kDa protein, malate dehydrogenase, succinyl-CoA synthetase alpha subunit, an amino acid ABC type transporter, Leu/ Ile/ Val-binding protein precursor, and ClpP. The remaining eight proteins had N-terminal sequences lacking similarity to existing database entries. Thus, the 2-D PAGE analysis provided a convenient first approach in the characterization of immunogenic proteins.
    Additional Material: 4 Ill.
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  • 8
    ISSN: 0173-0835
    Keywords: Yeast ; Saccharomyces cerevisiae ; Two-dimensional polyacrylamide gel electrophoresis ; Protein database ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: With the systematic sequencing of the yeast genome, yeast biology has entered a new era where novel challenges have to be faced. One challenge is the identification of the function of the several hundred novel genes discovered by genome sequencing. Another is to understand how all yeast genes act in concert to ensure and maintain cell organization. Two-dimensional (2-D) gel electrophoresis is the technique of choice to take up these challenges because it provides the opportunity of obtaining an overall view of genome expression. In prospect of these studies we have undertaken the construction of a yeast 2-D gel protein database that contains information on polypeptides of the yeast protein map. In this paper we report the information presently contained in this database. The reported information includes the identification of 250 protein spots and the characterization of polypeptides corresponding to N-terminal acetylated proteins, mitochondrial proteins, glucose-repressed proteins, heat shock induced proteins and proteins encoded by intron-containing genes. In all, 600 spots are annotated. These data can be accessed on the Yeast Protein Map server through the World Wide Web network.
    Additional Material: 7 Ill.
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  • 9
    ISSN: 0173-0835
    Keywords: Phagosome maturation ; Endosomes ; Membrane traffic ; Two-dimensional polyacrylamide gel electrophoresis ; Phagolysosome biogenesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Cells perform their multiple functions with the aid of a series of distinct membrane organelles. In the last years, many of these compartments have been isolated, purified, and extensively studied. The major roles of each organelle in the cell are well understood. However, most of the molecular basis by which they perform their functions is poorly known. The recent identification and study of a handful of proteins associated with endovacuolar compartments has had a major impact on the understanding of the molecular details of organelle functions even though two-dimensional (2-D) gel analysis indicates that hundreds of proteins are typically associated with a complex organelle. This shows that many details and surprises are still to come for cell biologists. In the present study, we have analyzed and compared different organelles of the endocytic and phagocytic apparatus using 2-D gel electrophoresis.
    Additional Material: 4 Ill.
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  • 10
    ISSN: 0173-0835
    Keywords: Sample application ; Two-dimensional polyacrylamide gel electrophoresis ; Immobilized pH gradient ; Protein characterization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple and inexpensive methacrylate rehydration chamber was built to accomodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration. For rehydration, commercially available or laboratory-made strips were positioned in the grooves with the gel in contact with 500 μL of sample for 6 h or overnight. This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.
    Additional Material: 4 Ill.
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