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  • 1
    Electronic Resource
    Electronic Resource
    An immunological study of corrinoid proteins from bacteria revealed homologous antigenic determinants of a soluble corrinoid-dependent methyltransferase and corrinoid-containing membrane proteins from Methanobacterium species (1990)
    Springer
    Archives of microbiology 155 (1990), S. 28-34 
    Details
    ISSN: 1432-072X
    Keywords: Methylmalonyl-CoA mutase ; 2-Methyleneglutarate mutase ; Carbon monoxide dehydrogenase ; Adenosylcobalamin ; Methyl-vitamin B12 ; Methanobacterium thermoautotrophicum ; Propionibacterium shermanii
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The hypothesis of common epitopes in corrinoid-dependent enzymes was tested by a monospecific polyclonal antiserum against the 33 kDa corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum Marburg. Cross-reaction was detected with the 33 kDa and the 31 kDa subunits of the corrinoid-containing enriched 5-methyl-H4MPT: 5-hydroxybenzimidazolyl cobamide methyltransferase from the cytoplasmic fraction and a 33 kDa protein from the membrane fraction of Methanobacterium thermoauto-trophicum ΔH. This indicates that both proteins have similar antigenic determinants and that they may have similar function as methyltransfer proteins. Also a soluble 20 kDa protein of yet unknown function from Clostridium barkeri cross-reacted with the antiserum. No cross-reactions were observed with the purified corrinoid-containing 2-methyleneglutarate mutase from C. barkeri, the corrinoid/iron-sulfur protein from C. thermoaceticum, the carbon monoxide dehydrogenases from C. thermoaceticum and Methanothrix soehngenii, and the corrinoid-binding protein intrinsic factor from porcine gastric mucosa. Also cell extracts from the corrinoid-rich bacteria Sporomusa ovata, Methanolobus tindarius, Chloroflexus aurantiacus, Propionibacterium shermanii, the membrane fraction and the cytoplasmic fraction of Methanococcus voltae or extracts from human liver, contained no antibody combining sites others than with the preimmunological serum. These findings indicate, that many corrinoid-containing proteins from bacteria have no common antigenic determinants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00291270
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  • 2
    Electronic Resource
    Electronic Resource
    Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources (1990)
    Weinheim : Wiley-Blackwell
    Electrophoresis 11 (1990), S. 581-588 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Purification and chemical characterization of protein may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60 % and 100 % were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60 % and 79 %. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patters, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150110709
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  • 3
    Electronic Resource
    Electronic Resource
    Combination of two-dimensional gel electrophoresis with microsequencing and amino acid composition analysis: Improvement of speed and sensitivity in protein characterization (1990)
    Weinheim : Wiley-Blackwell
    Electrophoresis 11 (1990), S. 554-561 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: High-resolution two-dimensional polyacrylamide electrophoresis (2-DE) is commonly used as an analytical approach to resolve and detect most of the numerous protein species of an organism. However, the isolation of microgram amounts of protein in a 2-DE spot in a form suitable for microsequence analysis and amino acid composition analysis is a key step in the chemical characterization of these proteins. With the development of chemically inert membranes it is now possible to retain proteins present in low quantities from the polyacrylamide matrix with high yields. The immobilized proteins are suitable for direct sequence analysis and amino acid composition analysis. The combination of protein chemical and electrophoretic techniques makes it possible to obtain chemical information from subpicomole quantities of protein, resulting in the availability of a new set of biologically important proteins for structural analysis. This paper summarizes the methods and strategies for the chemical protein analysis of 2-DE spots in our laboratory.
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150110705
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  • 4
    Electronic Resource
    Electronic Resource
    Structural characterization of blotting membranes and the influence of membrane parameters for electroblotting and subsequent amino acid sequence analysis of proteins (1993)
    Weinheim : Wiley-Blackwell
    Electrophoresis 14 (1993), S. 831-838 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Various blotting membranes were evaluated and correlated with the efficiency of electroblotting and the performance in the sequencing process. Structural parameters including specific surface area, pore size distribution, pore volumes, and permeabilities of different solvents lead to discrimination of the membranes relative to their accessible surfaces and membrane densities. Protein binding capacities as well as protein recoveries in electroblotting correlate with the specific surface areas. Almost quantitative retention of proteins during electroblotting from gels was obtained for membranes with a high specific surface area and narrow pores (Trans-Blot, Immobilon PSQ, Fluorotrans), whereas membranes with a relatively low specific surface area (Immobilon P, Glassybond) showed reduced recoveries of between 10-20% for the tested proteins. Initial yields and repetitive yields were compared for radioiodinated standard proteins that have been either electroblotted or loaded by direct adsorption. The results showed that the different permeabilities for solutions of the Edman chemistry have a major influence on initial yields. The glass fiber-based membranes with an extremely low flow restriction produce consistently high initial yields independent of the application mode of the protein (spotted or electroblotted) or the application of the membranes into the cartridge (discs or small pieces). In contrast, the polymeric membranes showed decreasing initial yields with increasing membrane density for spotted and electroblotted proteins. Yields varied considerably when the membranes were applied as discs into the cartridge. This effect could be minimized by cutting the membranes into piecese as small as possible, as demonstrated for electro-blotted proteins.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501401133
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  • 5
    Electronic Resource
    Electronic Resource
    Enhanced in situ gel digestion of electrophoretically separated proteins with automated peptide elution onto mini reversed-phase columns (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 899-906 
    Details
    ISSN: 0173-0835
    Keywords: In-gel digestion ; Automation ; Peptide elution ; In-line column adapter ; Protein sequencing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: An improved method for the generation and automated isolation of internal peptides by in situ gel digestion of electrophoretically separated proteins is described [1]. To enhance the sensitivity of the method, and to reduce the amount of sample handling steps, we have automated the extraction procedure of peptides after protein cleavage in a sodium dodecyl sulfate (SDS) gel matrix. The excised protein-containing polyacrylamide bands or spots are first minced to defined particles of about 30 μm. After in situ gel digestion, the gel slurry is transferred into a mini reversed-phase column-funnel assembly in the sample loading station of the Hewlett-Packard protein sequencer. Applying nitrogen pressure elutes peptides from the gel slurry onto the reversed-phse material. The mini reversed-phase column is then placed in an in-line column adapter and connected to a micropreparative high performance liquid chromatography (HPLC) column, where separation of the peptides under standard conditions is achieved. In the work described here complete digestions and excellent peptide recoveries allowed the generation of extensive internal sequence information from low picomole amounts of proteins. The method has been routinely applied in both laboratories for two yearsPart of the results were presented as a poster at the Protein Society Meeting 1994 in San Diego..
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170511
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  • 6
    Electronic Resource
    Electronic Resource
    Ultraviolet matrix assisted laser desorption ionization-mass spectrometry of electroblotted proteins (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 954-961 
    Details
    ISSN: 0173-0835
    Keywords: Mass determination ; Matrix-assisted laser desorption ionization-mass spectrometry ; Electroblotting ; Protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Direct mass spectrometric analysis of proteins electroblotted onto polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis is demonstrated by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) with a linear time-of-flight instrument, equipped with a nitrogen laser (337 nm). The blotted proteins were desorbed directly from the blotting membrane after incubation with sinapinic acid as matrix. Different commercially available membranes resulted in high quality protein signals for hydrophobic membranes exhibiting high specific surface areas (Immobilon PSQ or Trans-Blot) or for charged membranes (Immobilon CD). Systematic investigations with standard proteins were performed to compare standard preparation procedures for ultraviolet (UV) MALDI-MS on stainless steel sample stages and preparation of proteins immobilized onto membranes either by direct application from protein solutions (spotting) or by electrotransfer from gels (electroblotting). Aspects such as mass resolution, reproducibility from shot to shot and spot to spot, mass accuracy, and preservation of protein localization are addressed in this paper.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170518
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  • 7
    Electronic Resource
    Electronic Resource
    Towards a two-dimensional database of common human proteins (1992)
    Weinheim : Wiley-Blackwell
    Electrophoresis 13 (1992), S. 729-732 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A protein pattern of common human proteins was constructed by comparing the two-dimensional gel electrophoresis (2-DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2-DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2-DE protein map of only common human proteins was constructed as a basis for a new 2-DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501301156
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  • 8
    Electronic Resource
    Electronic Resource
    Mass spectrometric analysis of blotted proteins after gel electrophoretic separation by matrix-assisted laser desorption/ionization (1992)
    Weinheim : Wiley-Blackwell
    Electrophoresis 13 (1992), S. 664-665 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The molecular masses of electroblotted proteins were determined with a time-of-flight mass spectrometer by matrix-assisted laser desorption/ionization directly from blot membranes. Therefore standard proteins, separated by polyacrylamide gel electrophoresis, were electroblotted onto polyvinylidene difluoride or polyamide membranes by standard procedures. Pieces of membrane containing the protein of interest were soaked in matrix solution and analyzed in the mass spectrometer.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501301140
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  • 9
    Electronic Resource
    Electronic Resource
    Suppression effects in enzymatic peptide ladder sequencing using ultraviolet - matrix assisted laser desorption/ionization - mass spectrometry (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 1910-1919 
    Details
    ISSN: 0173-0835
    Keywords: Matrix assisted laser desorption ; ionization - mass spectrometry ; Suppression effects ; Enzymatic sequencing ; Peptide sequencing ; Ladder sequencing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The techniques of enzymatic and chemical peptide ladder sequencing, coupled with ultraviolet - matrix assisted laser desorption/ionization - mass spectrometry (UV-MALDI-MS) have been improving continuously in the last five years and have now become important tools for primary structure identification. In this work, signal suppression effects, appearing in UV-MALDI-MS (excitation 337 nm) of ladder peptides, were investigated using the 17-amino acid peptide dynorphin A. We show, with examples of simple “two-peptide” systems and more complex “multi-peptide” systems, that suppression effects do in fact exist. The magnitude of the observed suppression is strongly dependent upon both the nature and the amount of the suppressing peptide. Suppression behavior of individual ladder peptides was investigated on equimolar mixtures of ten ladder peptides. Significant signal suppression was recorded for all ladder peptides, with some of them being approximately 170 times lower in signal intensity than the pure, i.e., unsuppressed peptide at the same concentration. For the investigated system  -  dynorphin A, 4-hydroxy-α-cyanocinnamic acid (4-HCCA) matrix, UV excitation  -  a correlation between the extent of suppression and an intractable combination of peptide hydrophobicity and the presence of several basic amino acids can be seen.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150191109
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  • 10
    Electronic Resource
    Electronic Resource
    Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis (1988)
    Weinheim : Wiley-Blackwell
    Electrophoresis 9 (1988), S. 830-838 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low μg range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensionalelectrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150091208
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