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  • Biochemistry and Biotechnology  (16,218)
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  • 1
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 134-140 
    ISSN: 0952-3499
    Keywords: affinophoresis ; affinophore ; affinity probe capillary electrophoresis ; pea lectin ; concanavalin A ; laser-induced fluorescence detection ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Combination of capillary electrophoresis and bioaffinity interaction gave rise to powerful research tools for analyzing molecular recognition. They take advantages of the electrophoretic behavior of the complex formed between a target biomolecule and a specially designed mobile ligand molecule (affinophore or affinity probe), and enable detection of complex formation, determination of the equilibrium constants and stoichiometry, etc. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 2
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 128-133 
    ISSN: 0952-3499
    Keywords: immunoglobulin purification ; combinatorial chemistry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A synthetic ligand [TG19318], able to mimic protein A in the recognition of the immunoglobulin Fc portion, has been previously identified in our laboratory through the synthesis and screening of multimeric combinatorial peptide libraries. In this study we have fully characterized its applicability in affinity chromatography for the downstream processing of antibodies, examining the specificity and selectivity for polyclonal and monoclonal immunoglobulins derived from different sources. Ligand specificity was broader than protein A, since IgG deriving from human, cow, horse, pig, mouse, rat, rabbit, goat and sheep sera, IgY obtained from egg yolk, and IgM, IgA and IgE were efficiently purified on TG19318 affinity columns. Adsorbed antibodies were conveniently eluted by a buffer change to 0.1 M acetic acid or 0.1 M sodium bicarbonate pH 9, with full retention of immunological properties. Monoclonal antibodies deriving from cell culture supernatants or ascitic fluids were also conveniently purified on TG19318 affinity columns, even from very diluted samples. The affinity constant for the TG19318-IgG interaction was 0.3 µM, as determined by optical biosensor measurements. Under optimized conditions, antibody purity after affinity purification was close to 95%, as determined by densitometric scanning of SDS-PAGE gels of purified fractions, and maximal column capacity reached 25 mg Ig/ml support. In vivo toxicity studies in mice indicated a ligand oral toxicity greater than 2000 mg kg-1 while intravenous toxicity was close to 150 mg kg-1. Validation of antibody affinity purification processes for therapeutic use, a very complex, laborious and costly procedure, is going to be simplified by the use of TG19318, which could reduce considerably the presence of biological contaminants in the purified preparation, a very recurrent problem when using recombinant or extractive biomolecules as affinity ligands. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 3
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 149-150 
    ISSN: 0952-3499
    Keywords: electrophoresis ; preparative electrophoresis ; multichannel flow electrophoresis ; proteins ; enzymes ; antibodies ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Multichannel flow electrophoresis (MFE) is a newly developed method for continuous separation of biological products at a preparative scale, In this short survey, the application of MFE in the separation of proteins, enzymes and antibodies are overviewed. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 4
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 141-148 
    ISSN: 0952-3499
    Keywords: capillary electrophoresis ; binding studies ; affinity ; binding constants ; heparin ; amyloid P component ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Molecular recognition may be characterized both qualitatively and quantitatively by electrophoretic methods if complexed molecules differ in electrophoretic mobility from unbound ones. The use of capillary zone electrophoresis (CE) for the characterization of affinity interactions is advantageous because of the high resolution, reproducibility and wide applicability of the technique and because of the mild conditions, i.e. physiological buffers without additions of organics or detergents, that are often sufficient for highly efficient separations. CE gives the ability to characterize binding between small amounts of unlabelled reactants in solution, has few requirements for special chacteristics of the interacting molecules and is also applicable to the study of interactions of individual components in mixtures, as detection of binding and analytical separation are achieved in one step. This is unique compared with other techniques for the study of non-covalent interactions. The advantages and disadvantages of using CE to demonstrate molecular interactions, to screen for specific ligand binding in complex mixtures and to calculate binding constants will be discussed. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 5
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 151-156 
    ISSN: 0952-3499
    Keywords: electrophoretic affinity chromatography ; affinity chromatography ; electrophoresis ; human serum albumin ; Blue Sepharose Fast Flow ; multicompartment electrolyser ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A new method for preparative-scale separation of biomolecules, electrophoretic affinity chromatography (EAC), is proposed in this paper. Separation by EAC is carried out in a long and ribbon-like multicompartment electrolyser separated by membranes, in which the two central compartments are used for packing the gel matrix and for sample loading respectively. Next to the central compartments are the elution compartments and electrode compartments. The electric field is applied perpendicular to the fluid flow in the compartments. Adsorption and desorption steps may both be carried out in the presence of an electric field, which transports the target components into the gel compartment for adsorption and the impurities into the elution compartments for washing. After the adsorption step an elution solution is introduced and the product is released from the gel matrix and washed out. Separation of human serum albumin (HSA) from human serum gives HSA product of high purity, as demonstrated by isoelectric focusing analysis. The characteristics of electrophoretic binding of HSA on Blue Sepharose Fast Flow are examined. The preliminary results show that this new method has advantages in terms of high rate of mass transfer and ease of scaling up, which are of particular interest when large-scale separation of biomolecules is considered. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 6
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 255-260 
    ISSN: 0952-3499
    Keywords: displacement ; dye-ligand chromatography ; polymer ; poly(ethylene imine) ; poly(N-vinyl pyrrolidone) ; poly(N-vinyl caprolacatam) ; protein purification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Displacement chromatography was demonstrated to perform separations efficiently under mass-overloaded conditions, offering advantages such as increased product recovery and purity, superior resolving power, and concentration and purification in a single processing step. The use of water-soluble polymers for protein displacement in dye-ligand chromatography was initiated in our laboratory. The polymers for displacement were selected using difference spectroscopy to monitor their interactions with a dye-ligand in solution. Non-charged polymers such as poly(N-vinyl pyrrolidone) and poly(N-vinyl caprolactam) efficiently displaced lactate dehydrogenase from porcine muscle from a Blue Sepahrose column. The latter polymer, being thermosensitive, could be easily removed from the eluate and recovered by precipitation at 45 °C and low-speed centrifugation. The positively charged polymer poly(ethylene imine) proved to be an even more efficient displacer. The dye-ligand column could be regenerated after application of displacer either by washing with a solution of the soluble ligand Cibacron Blue (in the case of non-charged polymers) or by washing with highly alkaline solutions containing polyanions (in the case of poly(ethylene imine)) The latter formed a soluble complex with poly(ethylene imine) and stripped the column from the polymer. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 7
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 261-262 
    ISSN: 0952-3499
    Keywords: aza-arenophilic supports ; IgG purification ; reversible adsorption of enzymes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A number of synthetic aza-arenophilic gels have been synthesized from Sepharose CL-4B, 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine. These show high binding capacities for immunoglobulins and enzymes. Under high-salt buffer binding conditions, IgG can be effectively eluted, essentially free of contamination by BSA, using acidic conditions (pH 2.5) or phosphate buffers (pH 7.4) containing nucleophiles. Enzymes can also be readily adsorbed and desorbed. Thus these gels can be reused as supports for the immobilization of enzymes. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 8
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 28-31 
    ISSN: 0952-3499
    Keywords: molecular recognition ; solvatochromic parameter ; extraction ; phenobarbital ; solvent ; selectivity ; yield ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The extraction efficiency for organic molecules using synthetic receptors is highly dependent on solvent properties. Solvent influences the partitioning of both the desired compound and the interfering species. Solvent also influences the solubility of the receptor and its affinity for the substrate. Therefore the free energy involved in extraction can be optimized by using a carefully selected solvent. In this paper we demonstrate the use of a solvatochromic model to predict the influence of solvent dipolarity, H-bond acidity and H-bond basicity on selectivity and yield of phenobarbital extraction. We also used this method to estimate the purity and yield of phenobarbital extraction in 12 poly(vinyl chloride) plasticizers and solvents. This approach can be generalized for assisting the selection of optimal solvent and provide insight into the rational design of solvent and receptor for industrial extractions. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 9
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 25-27 
    ISSN: 0952-3499
    Keywords: artificial chaperone ; polyelectrolyte complexes ; glyceraldehyde-3-phosphate dehydrogenase ; enzyme inactivation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies, which bind specifically inactive dimers of glyceraldehyde-3-phosphate dehydrogenase but not native tetramers, were coupled covalently to poly(methacrylic acid). The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of polycation, poly-(N-ethyl-4-vinylpyridinium bromide), resulted in a significant increase in the specific activity of the enzyme. Copyright © 1998 John Wiley & Sons, Ltd
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  • 10
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 32-39 
    ISSN: 0952-3499
    Keywords: serine proteases ; trypsins ; subtilisins ; serine protease inhibitors ; IMAC ; histidine mapping ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The immobilized metal ion affinity (IMA) interaction of different serine proteases, namely porcine and bovine trypsins and BPN' and Carlsberg subtilisins, was studied on Sepharose-IDA-CuII. Both trypsins were resolved into their different subspecies, whereas the subtilisins appeared as only one species. The use of diethyl pyrocarbonate-modified enzymes demonstrated the contribution of histidine(s) as the sole interacting site(s). The use of different peptidic and chemical inhibitors complexed to the enzymes confirmed the contribution of histidine(s) as the interacting site(s) and further resulted in different chromatographic patterns for the free and complexed serine proteases. Comparison of the chromatographic data for each enzyme with the accessible surface area calculation by molecular modelling on the available crystallographic structure allowed us to hypothesize a map of the surface-accessible histidine on each enzyme. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 11
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 40-48 
    ISSN: 0952-3499
    Keywords: autoimmune disease ; Sjögren's syndrome ; lacrimal gland ; membrane traffic ; endocytosis ; protein sorting ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: CD4 T cell antigen recognition requires presentation by major histocompatibility complex Class II molecules (MHC II). B cell surface immunoglobulins recognize antigens independently of MHC II, but activation typically requires CD4 cell cytokines as accessory signals. Plasma membrane-endomembrane traffic in lacrimal gland acinar cells, targets of autoimmune activity in Sjögren's syndrome, may satisfy both requirements. The Golgi protein galactosyltransferase and the lysosomal proteins cathepsin B and cathepsin D appear at the plasma membranes during sustained secretomotor stimulation. The RNA transcription termination factor La, a frequent target of Sjögren's autoantibodies, appears in the acinar cell cytoplasm and plasma membranes during viral infection and during in vitro exposure to cytokines. MHC II cycle through endomembrane compartments which contain La, galactosyltransferase, cathepsin B and cathepsin D and which are sites of proteolysis. This traffic may permit trilateral interactions in which B cells recognize autoantigens at the surface membranes, CD4 T cells recognize peptides presented by MHC II, B cells provide accessory signals to CD4 T cells, and CD4 T cells provide cytokines that activate B cells. Acinar cells stimulate lymphocyte proliferation in autologous mixed cell reactions, confirming that they are capable of provoking autoimmune responses. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 12
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 157-162 
    ISSN: 0952-3499
    Keywords: capillary electrophoresis ; thermal field flow fractionation ; hyphenation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The possibility was considered to use the transverse thermophoresis of analytes in the capillary for capillary electrophoresis (CE) to control the separation process, decrease the peak width due to thermal effects and provide new separation parameters in CE. As the examination has shown, in non-aqueous buffers the Joule heating in the capillary for CE can provide transverse temperature gradients comparable with the temperature gradients in conventional devices for thermal field flow Fractionation (ThFFF). It was proposed to use the non-uniform velocity profile of analytes caused by the transverse temperature gradient and the temperature dependence of the buffer viscosity for the FFF-like separation of analytes besides CE separation. The expressions for the peak parameters have been derived, where the non-uniform transverse analyte concentration distribution due to the thermophoresis is taken into account, and the possibilities based on FFF-CE principles are discussed. As possible objects of this hyphenated technique, macromolecules and particles are considered. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 13
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 163-167 
    ISSN: 0952-3499
    Keywords: biosensors ; BIAcore ; epitope mapping ; thermodynamics ; concentration determination ; diagnostic reagents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The introduction in 1990 of a new biosensor technology based on surface plasmon resonance has revolutionized the measurement of antigen-antibody binding interactions. In this technique, one of the interacting partners is immobilized on a sensor chip and the binding of the other is followed by the increase in refractive index caused by the mass of bound species. The following immunochemical applications of this new technology will be described: (1) functional mapping of epitopes and paratopes by mutagenesis; (2) analysis of the thermodynamic parameters of the interaction; (3) measurement of the concentration of biogically active molecules; (4) selection of diagnostic probes. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 14
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 168-174 
    ISSN: 0952-3499
    Keywords: hologram ; poly(vinyl alcohol) ; protease ; trypsin ; poly(lysine) ; rational design ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A new silver halide-containing holographic recording material has been designed and developed specifically for holographic chemical sensors. The hologram enables very small volume changes to be measured in a polymer layer throughout which the hologram is located. The holographic film is based on a fine-grain silver bromide emulsion suspended in a poly(vinyl alcohol) matrix crosslinked with Cr(III) ions. Crosslinking gives the material sufficient spatial integrity to allow a holographic image to be recorded, while maintaining adequate porosity and elasticity of the polymer matrix for sensing applications. The new material has been characterized with respect to its response to pH and compared with a traditional gelatin holographic film. The response to some ions and small molecules typically found in analytical samples has also been measured. Functional groups introduced covalently into the poly(vinyl alcohol) matrix transform the base matrix into a pH-responsive polymer with predictable swelling properties and which can be further derivatized to incorporate specific ligands. A rationally designed holographic sensor for trypsin has been developed from chemically synthesized artificial polymers. A trypsin substrate, the poly(amino acid) poly(L-lysine), was incorporated into poly(vinyl alcohol) holograms to create a ‘designed’ holographic material which was degraded in a concentration-dependent manner by trypsin. Extensions of this approach to other hydrolytic enzymes are briefly discussed. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 15
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 178-181 
    ISSN: 0952-3499
    Keywords: immunoelectrode ; amperometry ; atrazine ; redox polymers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An amperometric immunosensor for the detection of the herbicide atrazine has been developed. A redox polymer PVPOs(bpy)2Cl was co-immobilized with the specific antibody on the surface of the electrode by crosslinking with PEGDGE to form an electron-conducting hydrogel. In a competitive assay the occurrence of the antibody-antigen reaction on the surface of the sensing film was detected through the ‘electrical wiring’ of the redox centres of antigen-labelled horseradish peroxidase and the electrode surface in the presence of H2O2 at 0.1 V (vsAg/AgCl). Copyright © 1998 John Wiley & Sons, Ltd.
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  • 16
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 175-177 
    ISSN: 0952-3499
    Keywords: immunosensor ; capacitance ; antibody-antigen affinity interactions ; Si-SiO2-Si3N4. ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: There is widespread interest in capacitance immunosensor systems which directly detect antigen binding to immobilized antibody. Our system comprises an active biolayer of antibodies bound to a silicon-silicon dioxide-silicon nitride (Si-SiO2-Si3N4) surface. As with other groups, our system initially gave poorly reproducible responses on addition of antigen. We mechanically degraded the Si-SiO2-Si3N4 surface, and the responses on addition of transferrin were monitored. The mechanical degradation allowed the affinity reaction to be ‘seen’ capacitively. Once the system was established, a comparison of capture antibodies was performed to establish the most effective biolayer. Three affinity reactions were examined: (a) 1D2A4, monoclonal antibody (mAb) to human transferrin, as the capture layer; (b) polyclonal goat anti-human transferrin antibody (PcAb) as the capture layer; and (c) 1D2A4 with transferrin (Tf) prebound as the capture layer. There was no response to addition of transferrin where 1D2A4 was the capture layer. Addition of transferrin when the polyclonal antibody was used as the primary layer resulted in a drop in measured capacitance. Addition of goat anti-human transferrin antibody to a device with 1D2A4 plus transferrin as the capture layer also resulted in a measured capacitance decrease. There is a difference in dielectric/blocking effectiveness between the monoclonal and polyclonal antibodies. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 17
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 191-193 
    ISSN: 0952-3499
    Keywords: Surface plasmon resonance ; low-molecular-weight heparin ; oligosaccharides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide-protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin™) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore™), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 18
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    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 188-190 
    ISSN: 0952-3499
    Keywords: weak affinity ; surface plasmon resonance ; carbohydrate ; antibody-antigen interactions ; hook effect ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Interactions between the immobilized weak-affinity monoclonal IgG antibody 39.5, which is specific for the glucose-α1,4-glucose motif, and various oligosaccharides were studied with surface plasmon resonance technology. The antibody was immobilized at high levels on the surface of the sensor chip and different concentrations of the analytes were injected at 25 and 40 °C. The 39.5 antibody exhibited specific binding to maltose, tetraglucose and maltotriose, with dissociation constants Kd in the range from 0.07 mM (25 °C) to 1.0 mM (40 °C). Association and dissociation rate constants (ka and kd) were rapid and baseline was obtained almost immediately after the end of each antigen injection. This excluded the need for a regeneration step but also made calculation of the kinetic values impossible. Owing to the weak affinity and the small size of the analytes (〈1000 Da), a careful design of control surfaces is demanded to exclude artefactual results. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 19
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    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 185-187 
    ISSN: 0952-3499
    Keywords: polyacrolein latex immunoreagent ; immunoradiometric assay ; ferritin ; particle agglutination assay ; immunofiltration dot assay ; S. pyogenes polysaccharide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Immunoreagents based on polymer dispersions consisting of unimodal polyacrolein (PAL) microspheres with diameters in the range 0.3-2.0 µm have been prepared and evaluated by various immunoassay techniques such as immunoradiometric assay of ferritin and microtitre particle agglutination and immunofiltration dot assay of group-specific polysaccharide of S. pyogenes (A-PS) in comparison with conventional carriers and methods. The antibodies were covalently or indirectly bound to the PAL. The coupled antibodies to ferritin retained a high average affinity (Ka = 4.5 × 109 M-1). In comparison with microcrystalline cellulose-based immunosorbent, more than an order-of-magnitude lower amount of PAL-IgG was necessary for the analysis of ferritin. Use of PAL-IgG gave a higher sensitivity of assay with a detection limit of 0.7 × 10-13 M l-1 and a wider concentration range of antigen detection (about four orders of magnitude) without manifestation of the high-dose hook effect. Particle agglutination assay of A-PS in microtitre plate was shown to be a simple, demonstrative and highly sensitive one-step analytical method with a detection limit of 0.05 ng A-PS/ml or 104 cells/ml. The sensitivity of immunofiltration assay using both enzyme and latex markers was shown to be approximately the same (50 ng A-PS/ml) and the duration of the assay was 3-5 min. No cross-reactions of latex conjugates with non-A Streptococcus cell lysates were observed. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 20
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    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 194-199 
    ISSN: 0952-3499
    Keywords: evanescent wave biosensor ; IAsys ; kinetics ; equilibria ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Trends in the analysis of molecular recognition using the IAsys evanescent wave biosensor are outlined. Diversification of sensor surface chemistry, an open cuvette format and the advent of robotics controlled by intelligent software are widening the range and throughput of applications. Analyses of binding and dissociation are now carried out across a wide spectrum of biomolecules, including protein, nucleic acid, carbohydrate and lipid. Determinations are obtained from a range of experimental formats. These include qualitative ‘yes/no’ screening assays, through semi quantitative ranking of kinetic association, dissociation and equilibrium constants for a family of binding partners, to deriving constants comparable with those which would be obtained in free solution. A dependence of the initial rate of biomolecular association on concentration allows analyte concentration to be measured - an increasingly common application class. This is often employed in situations where a rapid determination is required. The ability to recover bound analyte from the sensor surface in sufficient amounts for subsequent characterization is opening up new routes to the parallel analysis of structure and function. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 21
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    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 204-210 
    ISSN: 0952-3499
    Keywords: Kinetics ; thermodynamics ; protein ; enthalpy ; entropy ; equilibrium ; transition state ; BIACORE ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A methodology using biosensor technology for combined kinetic and thermodynamic analysis of biomolecular interactions is described. Rate and affinity constants are determined with BIAcore. Thermodynamics parameters, changes in free energy, enthalpy and entropy, are evaluated from equilibrium data and by using rate constants and transition state theory. The methodology using van't Hoff theory gives complementary information to microcalorimetry, since only the direct binding is measured with BIAcore whereas microcalorimetry measures all components, including e.g. hydration effects. Furthermore, BIAcore gives possibilities to gain new information by thermodynamic analysis of the rate constants. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 22
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    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 211-216 
    ISSN: 0952-3499
    Keywords: metal chelate affinity precipitation ; N-isopropylacrylamide ; 1-vinylimidazole ; iminodiacetic acid ; thermoprecipitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Affinity precipitation is fast emerging as a successful technique for the purification of proteins which can be introduced at an early stage of downstream processing. The technique applies the use of reversibly soluble-insoluble polymers which have either natural or synthetic origin. Apart from the successful use of some natural polymers, such as chitosan and alginate, the vast application of the technique depends upon the design of efficient synthetic polymers. In this laboratory, N-isopropylacrylamide (NIPAM) copolymers have been developed for metal chelate affinity precipitation of proteins. The copolymers of 1-vinylimidazole (VI) and iminodiacetic acid (IDA) with NIPAM were synthesized. The copolymers were thoroughly characterized with a view to designing an efficient soluble-insoluble polymer for metal chelate affinity precipitation of proteins. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 23
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    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 222-230 
    ISSN: 0952-3499
    Keywords: lipopolysaccharides ; protein contamination ; endotoxion removal ; selective adsorption ; polycationic ligands ; membrane adsorbers ; affinity membranes ; endotoxin-specific sorbents ; microfiltration membranes ; flat-sheet membranes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: For the removal of remaining amounts of endotoxin, sorbents with high selectivity for endotoxin are required. Typically, particulate sorbents with positively charged ligands, such as histidine, polymyxin B, poly-L-lysine and poly(ethyleneimine) (PEI), display moderate to high removal efficiencies in an environment of low ionic strength. It was found that polycationic ligands are most suitable to meet an endotoxin concentration which is below the threshold level required for parenteralia. Furthermore, protein recoveries close to 100% are obtained if the decontamination is performed at a pH close to the pI of acidic proteins. The high selectivity is probably caused by complexation of the polycationic ligand with the polyanionic endotoxin, leading to interactions with KD 〈 10-9 M using PEI and assuming Mr = 10 kDa for monomeric endotoxin; with BSA the same ligand reveals only KD = 4 × 10-6 M. Using polymer-coated microfiltration membranes, immobilization of positively charged ligands leads to membrane adsorbers which are generally superior to chromatographic adsorbers and allow faster processing. Since immobilization takes place at polymer chains, low-molecular-weight ligands mainly add positive charges to the hydrophilic polymer. Consequently, membrane adsorbers with low-molecular-weight ligands, even DEAE, demonstrate similar selectivity to PEI or poly-L-lysine. Copyright © 1998 John Wiley & Sons, Ltd.
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    Journal of Molecular Recognition 11 (1998), S. 240-242 
    ISSN: 0952-3499
    Keywords: monoclonal antibodies ; purification ; affinity precipitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A simple procedure for the purification of an IgG-type monoclonal antibody by affinity precipitation using Eudragit S-100 is presented. The ligand, a microbial lipase previously used as antigen, was coupled to the polymer at a concentration of 40 mg lipase/g Eudragit. This macroligand was reversibly precipitated by manipulating the pH at values higher and lower than 4.8. The effects of polymer concentration and dilution of hybridoma culture supernatant on the overall precipitation process were evaluated. The best purification factor was achieved with a polymer concentration of 0.1% (w/v) and a supernatant dilution of 1:3. The preliminary studies reported here enabled the purification of a monoclonal antibody in one step with an activity yield (by ELISA) of 50%- 55% and a purification factor of ca 6. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 25
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    Journal of Molecular Recognition 11 (1998), S. 243-246 
    ISSN: 0952-3499
    Keywords: immunoglobulin A purification ; protein A mimetic ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We reported previously that TG19318, a synthetic ligand deduced from the screening of combinatorial libraries, displays specific and selective recognition properties for immunoglobulins of the G class and can be used conveniently for affinity chromatography purification of monoclonal and polyclonal antibodies. In this study we have extended the ligand characterization, examining its ability to bind IgA from cell culture supernatants and from IgG-deprived serum. Affinity columns prepared by immobilizing TG19318 on Sepharose allowed convenient one-step purification of monoclonal IgA directly from crude feedstocks, in high yield and with full recovery of immunoreactivity. Optimal column adsorption occurred with phosphate buffer at neutral pH, while elution of adsorbed IgA could be accomplished by a buffer pH change to acidic or basic conditions. Column capacity was close to 7 mg IgA/ml support. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 26
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    Journal of Molecular Recognition 11 (1998), S. 231-235 
    ISSN: 0952-3499
    Keywords: product removal ; γ-decalactone ; cyclodextrin ; castor oil ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In situ product removal (ISPR) involves actions taken for the fast removal of a product from the producing cell. ISPR is implemented to improve yield and productivity via minimization of product inhibition, minimization of product losses due to degradation or evaporation, and reduction of the number of subsequent downstream processing steps. Here we describe the implementation of affinity-based, specific ISPR as a crucial component of an integrative approach to problems associated with the biocatalytic production of a product exhibiting poor water solubility from an oily, water-insoluble precursor. Our integrative ISPR-based approach consists of co-immobilization of the oily substrate emulsion and the biocatalyst within bilayered alginate beads. A particulate-specific adsorbent, exhibiting high binding capacity of the product, is suspended in the reaction medium with periodical replacements. According to this approach, ISPR implementation is expected to shift the equilibration of product distribution between the co-immobilized oily substrate and the outer medium via specific product immobilization onto the added adsorbent. The product may subsequently be readily recovered via single-step final purification. This integrative approach was successfully demonstrated by the affinity-based ISPR of γ-decalactone (4-decanolide). γ-Decalactone was produced from castor oil via its β-oxidation by the filamentous fungus Tyromyces sambuceus, co-immobilized with emulsified substrate within bilayered alginate beads. Product immobilization onto medium-suspended epichlorohydrin-crosslinked β-cyclodextrin resulted in higher yield and easy pure product recovery. Copyright © 1998 John Wiley & Sons, Ltd.
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    Journal of Molecular Recognition 11 (1998), S. 236-239 
    ISSN: 0952-3499
    Keywords: affinity precipitation ; dynamic laser light scattering ; Eudragit ; concanavalin A ; complex formation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The final outcome of an affinity precipitation process will depend upon the efficiency of each individual stage involved: the formation of initial affinity complexes, the build-up of a precipitate and the elution of the target protein. Investigations on the first stage were done in this study utilizing a model system. The target protein was the lectin concanavalin A (Con A). Eudragit S-100, a reversibly soluble/insoluble polymer consisting of methyl methacrylate and methacrylic acid, to which the affinity ligand p-aminophenyl-α-D-glucopyranoside was coupled, served as the bifunctional ligand (ligand-Eudragit). Owing to the tetrameric structure of Con A, where each subunit has the ability to bind one sugar moiety, and to the multivalency of ligand-Eudragit, a network was formed between the Con A and ligand-Eudragit. It was possible to detect the initial soluble complexes formed by dynamic laser light scattering (DLLS) long before any precipitate could be analysed by transmittance measurements. The rate of complex formation was highly dependent on the ratio between lectin and ligand-Eudragit. It was further shown that the system did not reach equilibrium within the 110 min studied. When the complex formation was studied in the presence of glucose, the build-up rate was decreased to different degrees depending on the sugar concentration used. At high glucose concentrations the complex formation was completely inhibited. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 28
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    Electrophoresis 19 (1998), S. 333-343 
    ISSN: 0173-0835
    Keywords: Breast ; Tumor ; Protein ; Two-dimensional polyacrylamide gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: High-resolution two-dimensional gel electrophoresis (2-DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2-DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent (56/727) of the consistently detected proteins were found in significantly (P 〈 0.001) variable levels among the cell lines. Eight proteins present in normal cultured breast epithelial cells were not detected in any of the tumor cell lines. We identified a subset of the differentially expressed proteins using a combination of immunostaining, protein sequencing, comigration, and subcellular fractionation. These identified proteins include the intermediate filament components vimentin and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27 and hsp60 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Many of the differentially expressed proteins we identified have roles in cellular proliferation and differentiation, including annexin V, elongation initiation factor 5A, Rho GDP dissociation inhibitor, and prohibitin. We identified inosine-5-monophosphate dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells. These results expand the human breast epithelial cell protein database (http://www.anl.gov/CMB/PMG) which is being built to assist researchers with the identification of abnormal patterns of expression and pathways associated with malignancy.
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    Electrophoresis 19 (1998), S. 349-354 
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Immobilized pH gradients ; Thiourea ; Apolipoproteins ; Fat emulsions ; Particle size ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The two-dimensional polyacrylamide electrophoresis (2-D PAGE) of the plasma protein adsorption pattern previously established for polymeric nanoparticles was modified and transferred to oil in water emulsions for intravenous administration. The emulsions were incubated with citrated plasma, and separation from excess plasma was performed by centrifugation under optimized conditions: 15 000 g and three washing steps with 0.05 M phosphate buffer, pH 7.4. With this sample preparation, coalescence of droplets could be avoided and an unchanged surface area maintained, in addition the phosphate buffer minimized artificial IgG adsorption. Critical factors affecting sensitivity were contamination of the sample by oil residues and the use of thiourea in the immobilized pH gradients. Changes in the protein adsorption pattern caused by altered surface properties of the emulsion (i. e. adsorbed Poloxamer 407) were detectable when applying the optimized protocol. Knowledge of the protein adsorption patterns and their correlation to in vivo behavior opens the perspective for the development of intravenous emulsions for controlled drug delivery.
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    Electrophoresis 19 (1998), S. 364-364 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 31
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    Electrophoresis 19 (1998), S. 355-363 
    ISSN: 0173-0835
    Keywords: Rheumatoid arthritis ; Two-dimensional polyacrylamide gel electrophoresis ; Acute phase ; Piroxicam ; Tenidap ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional (2-D) gel analysis was used to examine differences in the levels of 19 plasma proteins: before and after an acute inflammatory reaction (parenteral typhoid vaccination) in normal subjects, between rheumatoid arthritis (RA) patients and normals and in RA patients treated with tenidap (120 mg) and piroxicam (20 mg). Typhoid vaccination increased levels of SAA, haptoglobin α1, haptoglobin α2, haptoglobin β and α1-anti-chymotrypsin but decreased transthyretin and apolipoprotein E. In RA patients, serum amyloid A (SAA), haptoglobin α2, haptoglobin β, α1-antichymotrypsin and C3 proactivator levels were elevated while apolipoprotein A-I, apolipoprotein A-IV, transthyretin, Gc-globulin, α2-HS glycoprotein, α2-macroglobulin and α1-B glycoprotein levels were decreased, compared to normals. Compared to piroxicam, tenidap lowered levels of α1-antiprotease and SAA but raised the levels of transthyretin, Gc-globulin, α2-HS-glycoprotein and α2-macroglobulin in RA patients. C-reactive protein (CRP) could not be quantified on 2-D gels but, When measured by rate nephelometry, levels were reduced after treatment with tenidap compared to piroxicam. The general pattern of the acute phase protein response to an acute inflammatory response to typhoid vaccination is similar to that in the chronic inflammatory condition, RA. The impact of tenidap on both positive and negative acute-phase proteins in RA patients could clearly be distinguished from that of piroxicam.
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    Electrophoresis 19 (1998) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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    Electrophoresis 19 (1998), S. i 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 34
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    Electrophoresis 19 (1998), S. 383-387 
    ISSN: 0173-0835
    Keywords: Separation theory ; Dynamic complexation capillary electrophoresis ; Capacity factor ; Multiple equilibria ; Higher order interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The analyte migration behavior in any chemical separation system can be described using a single equation that unifies all areas of separation science. This equation can be used in capillary electrophoresis (CE) to design separation systems, and to study interactions between analytes and additives. By using individual capacity factors for each analyte species present in the system, and with the knowledge of the characteristics of each interaction, one can predict the analyte migration behavior in complicated CE systems, including systems with multiple 1:1 interactions and/or higher order interactions.
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  • 35
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    Electrophoresis 19 (1998), S. 545-550 
    ISSN: 0173-0835
    Keywords: Pseudomonas aeruginosa ; Genetic diversity ; Origin of replication ; β-Lactamase gene ; Flagellin gene ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Sequence analysis of three representative gene loci, oriC, ampC and fliC, in 19 Pseudomonas aeruginosa strains revealed a low sequence diversity that does not correlate with the extensive diversity of P. aeruginosa habitats. Single point mutations lead to a mean sequence diversity of 0.40%, 0.38% and 0.59% for oriC, ampC and a-type fliC, respectively, but of only 0.05% for b-type flagellin genes. The analyzed genes encode highly conserved functions that are subject to strong selective pressure. The detected nucleotide substitutions of oriC, accumulating in a central 95 bp region, affect neither the putative DnaA binding sites nor the 13 bp direct repeats that presumably provide the sites to openoriC duplex DNA. Even in P. aeruginosa strain DSM 1128, which exhibits an unusually high sequence variability in several analyzed genes, the 9 bp and 13 bp motifs are conserved, reflecting their essential functional role in replication initiation. The two flagellin types, differing by 37-38% in their primary structure, exhibit pronounced structural and functional homology, as shown by alignment of flagellin variants by hydrophobicity index, probability of surface exposure, chain flexibility and antigenicity, and by cross-reactivity between both proteins using specific antisera. Five nonsynonymous nucleotide substitutions of ampC lead to β-lactamase variants that differ in recognition and turnover of substrate, as deduced from the three-dimensional structure of the highly homologous Enterobacter cloacae β-lactamase and confirmed by inhibition kinetics. The identified point mutations in the three genes are classified as selectively equivalent sequence variants indicating neutral genetic drift as a mechanism of molecular evolution in P. aeruginosa, rather than positive selection.
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    Electrophoresis 19 (1998), S. 573-576 
    ISSN: 0173-0835
    Keywords: Mycobacteria ; Genome organization ; Optical mapping ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Genome maps have been constructed for the mycobacterial pathogens Mycobacterium leprae and Mycobacterium tuberculosis, as well as for the attenuated vaccine strain Mycobacterium bovis BCG Pasteur. While the chromosomes of M. tuberculosis and M. bovis BCG Pasteur show extensive conservation at the grosslevel, comparison with M. leprae revealed a high degree of diversification, with a mosaic-like pattern apparent. The ordered libraries of M. tuberculosis and M. leprae produced during the course of these studies played a central role in the genome sequencing projects of these two bacilli, showing the utility of this approach for systematic sequencing of bacterial genomes.
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    Electrophoresis 19 (1998), S. 569-572 
    ISSN: 0173-0835
    Keywords: Salmonella ; Chromosome ; Pulsed field gel electrophoresis ; Recombination ; Rearrangements ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Early genetic studies showed conservation of gene order in the enteric bacteria. Two recent methods using pulsed field gel electrophoresis (PFGE) to determine the physical map of the genome are: (i) partial digestion with the endonuclease I-CeuI, which digests the DNA of bacteria in the rrn operon for rRNA (ribosomal RNA), thus establishing the “rrn genomic skeleton” (the size in kbp of the intervals between rRNA operons); (ii) analysis of XbaI and B1nI sites within Tn10 insertions in the chromosome. The order of I-CeuI fragments, which is ABCDEFG in S. typhimurium LT2 and E. coli K-12, was found to be conserved in most Salmonella species, most of which grow in many hosts (host-generalists). However, in S. typhi, S. paratyphi C, S. gallinarum, and S. pullorum, species which are host-specialized, these fragments are rearranged, due to homologous recombination between the rrn operons, resulting in translocations and inversions. Inversions and translocations not involving the rrn operons are seldom detected except for inversions over the TER (termination of replication) region. Additive genetic changes (due to lateral transfer resulting in insertion of nonhomologous DNA) have resulted in “loops” containing blocks of DNA which provide new genes to specific strains, thus driving rapid evolution of new traits.
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    Electrophoresis 19 (1998), S. 577-581 
    ISSN: 0173-0835
    Keywords: Neisseria gonorrhoeae ; Neisseria meningitidis ; Genome map ; Genomic rearrangements ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Current efforts to completely sequence the meningococcal and gonocococcal genomes raise the question whether the lessons learned from the sequenced strains may be safely extrapolated to other members of these species, or whether, in view of the fact that Neisseriae are highly recombinogenic and exhibit a high degree of horizontal intra- and interspecies genetic transfer, only clonespecific conclusions are valid. From the known physical and genetic maps of each of two gonococcal and meningococcal strains, it would appear that both species exhibit a species-specific conservation in their genetic organization while the interspecies comparison revealed several rearrangements, although still with a high overall similarity. However, these data contrast with other evidence suggesting intra-species rearrangements, such as the nonconserved I-CeulI macrorestriction patterns of different meningococcal and other neisserial strains. Since I-Ceul cuts within the 23S-rRNA sequence, the restriction pattern should give reliable information on the distribution of rrn loci in the neisserial genomes. Further studies are warranted to answer these questions.
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    Electrophoresis 19 (1998), S. 738-744 
    ISSN: 0173-0835
    Keywords: Cyclodextrin elektrokinetic chromatography ; Open tubular electrochromatography ; CHIRASIL-DEX ; Dual chiral recognition system ; Cyclodextrin zone electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The enantiomer separation of hexobarbital was investigated by open tubular electrochromatography (OTEC) using the chiral stationary phase (CSP) CHIRASIL-DEX (a permethylated β-cyclodextrin covalently linked to a dimethyl-polysiloxane) and by cyclodextrin-electrokinetic chromatogaphy (CD-EKC) using anionic β-cyclodextrin-sulfo-n-propyl ether (SPE-β-CD) and cationic β-cyclodextrin-2-hydroxy-3-trimethylammoniumpropyl ether chloride (HTAP-β-CD) added to the running buffer. By employing two chiral selectors, the enantiomer separation of hexobarbital was then studied simultaneously by OTEC with CHIRASIL-DEX and by CD-EKC with either SPE-β-CD or HTAP-β-CD in the dual chiral recognition mode. In conjunction with CHIRASIL-DEX, anionic SPE-β-CD decreased the chiral separation factor α due to compensation of enantioselectivity whereas the cationic additive HTAP-β-CD increased the chiral separation factor α due to enhancement of enantioselectivity. It is concluded that CHIRASIL-DEX imparts an opposite enantioselectivity to the enantiomers of hexobarbital as compared to the charged CDs SPE-β-CD and HTAP-β-CD. Unusual peak broadening phenomena are observed in the dual chiral recognition system comprised of CHIRASIL-DEX and HTAP-β-CD. The possible consequences of accidental dual chiral recognition systems caused by wall stacking effects of the mobile phase additives onto the inner surface of the capillary column are discussed.
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  • 40
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    Electrophoresis 19 (1998), S. 752-757 
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Double replica blotting ; Contact copy ; Immunoblotting ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The combination of two-dimensional electrophoresis (2-DE) and subsequent Western blot analysis with antibodies directed against common cellular proteins is a straightforward and reliable method to quickly generate fix points in a protein map. In order to assure high accuracy in the allocation of protein spots, two different replica blotting methods for semidry blotting devices were established. The first of the two was described by Johansson (Electrophoresis 1987, 8, 379-383). By systematically changing the direction of the blotting current, proteins were simultaneously transferred from one gel onto two membranes placed at both sides of the gel. However, several modifications of this method were necessary in order to use a semidry blotting device. The second method described here combines the standard blotting procedure with the generation of a ‘contact copy’ from the gel. Both systems offer the possibility to subject one membrane to antibody-mediated imaging, while the second membrane can be stained with highly sensitive total protein detection procedures. Protein identification is then carried out by comparing the signals on both matrices.
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    Electrophoresis 19 (1998), S. 745-751 
    ISSN: 0173-0835
    Keywords: Barley ; Chitinase ; Drechslera teres ; Glucanase ; Peroxidase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Molecular size and net charge of isoforms of pathogenesis-related (PR) chitinase, β-1,3-glucanase and peroxidase were studied in uninfected barley (Hordeum vulgare L., ν. Karat) leaves and in barley leaves infected with the pathogenic fungus Drechslera teres f. teres (Sacch.) Shoem. Molecular characteristics were determined by time-dependent polyacrylamide gradient gel electrophoresis under native conditions and by applying an extended version of the computer program MOL-MASS (Rothe, G. M., Weidmann, H., Electrophoresis 1991, 12, 703-709). Uninfected barley leaves contained predominantly one peroxidase isozyme but also three very weak peroxidases. Activities of all of these three peroxidases increased considerably after infection with Drechslera teres. The molecular masses of peroxidases 1 and 3 were estimated to be 38 ± 5 and 42 ± 7 kDa and their apparent valences at pH 8.4 were Z = 3.13 and 3.20, respectively. Amongst the chitinase isoforms, chitinase 1 and chitinase 2 appeared after infection, while chitinase 3 was also observed in uninfected leaves of barley. The molecular mass of chitinase 3 (31 ± 6 kDa; f/fo = 1.20) was larger than that of chitinase 1 (20 ± 2 kDa; f/fo = 1.04) and chitinase 2 (23 ± 3 kDa; f/fo = 1.06). The valence of constitutive chitinase 3 (Z = 1.44 ± 0.81) at pH 8.4 was lower than that of adaptive chitinase 1 (Z = 3.27 ± 1.02) and chitinase 2 (Z = 2.96 ± 1.38). Infection of barley leaves with Drechslera teres also induced the hydrolytic enzyme β-1,3-glucanase 1; β-1,3-glucanase 2 appeared in uninfected and in infected leaves. Constitutive β-1,3-glucanase 2 was smaller (molecular mass 19 ± kDa; f/fo = 1.05) than adaptive β-1,3-glucanase 1 (molecular mass 26 ± 4 kDa; f/fo = 1.07). The valence of adaptive β-1,3-glucanase 1 (Z = 9.58 ± 4.17) was approximately threefold that of β-1,3-glucanase 2 (Z = 2.80 ± 0.93).
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    Electrophoresis 19 (1998), S. 758-760 
    ISSN: 0173-0835
    Keywords: Membrane solubilization ; Thiourea ; Two-dimensional polyacrylamide gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The separation of membrane proteins by high-resolution two-dimensional electrophoresis was carried out. At high loads, these proteins are prone to precipitation, resulting in poor resolution. It is shown here that the use of thiourea, previously described for focusing in immobilized pH gradients, can be extended to conventional isoelectric focusing. As thiourea inhibits acrylamide polymerization, a modified photopolymerization system must be used. These modifications result in higher solubility of proteins during IEF, thereby increasing the resolution and capacity of the two-dimensional gels.
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    Electrophoresis 19 (1998), S. 761-766 
    ISSN: 0173-0835
    Keywords: Two-dimensional protein map ; Two-dimensional polyacrylamide gel electrophoresis ; Basic proteins ; Haemophilus influenzae ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Haemophilus influenzae is a bacterium of medical interest of which the entire genome has been sequenced. The proteome of the microorganism has been analyzed by two-dimensional gel electrophoresis, during which immobilized pH 3-10 gradient strips were used and approximately 300 proteins were identified. In order to detect additional, basic proteins, we analyzed the soluble protein fraction of H. influenzae and the proteins of fractions collected from affinity chromatography on heparin, by two-dimensional gel electrophoresis, using for the first-dimensional separation immobilized pH gradient strips comprising the pH region of 6-11. The protein spots were analyzed by matrix-assisted laser desorption ionization-mass spectrometry. One hundred and two proteins were identified, of which 58 were identified for the first time. A large percentage of the basic proteins represent nucleic acid binding and, in particular, ribosomal proteins. The locations of the identified basic proteins of H. influenzae are indicated in a two-dimensional map.
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    Electrophoresis 19 (1998), S. 776-781 
    ISSN: 0173-0835
    Keywords: C-reactive protein ; Paediatrics ; Serum amyloid A protein ; Two-dimensional polyacrylamide gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to analyze C-reactive - (CRP) and serum amyloid A protein (SAA) in infants and children. Five SAA isotypes were identified. CRP showed vertical streaking, and its optical density values correlated with immunoturbidimetric measurements. As evaluated by densitometry, both proteins showed an age-dependent variation. In more than 50% of the neonates, SAA was present in equal or higher amounts than CRP, and only SAA1α could be detected. In children, CRP was expressed in higher amounts than SAA, and both SAA1α and SAA2α were present. N-terminally modified forms of both isotypes were present regardless of age, including in premature infants. These results suggest that the overall synthesis of the gene products SAA1α and SAA2α is developmentally regulated, but at the same time that their N-terminal processing occurs independently of developmental factors. The presented data suggest that SAA has an important function in neonates, and that the role of SAA as an infection marker in this population should be investigated further.
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  • 45
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    Electrophoresis 19 (1998), S. 767-775 
    ISSN: 0173-0835
    Keywords: Charge shift ; Aging ; Monoclonal antibody ; Glycosylation ; Deamidation ; Two-dimensional polyacrylamide gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The heavy and light chains of IgG monoclonal antibodies (mAbs) can be shown to be heterogeneous, with respect to isoelectric points, when analyzed by two-dimensional electrophoresis (2-DE). The molecular basis for this charge heterogeneity has not been clearly defined but it has been suggested that it could be due, in part, to differences in glycosylation. To investigate this possibility we have compared the 2-DE pattern of glycosylated and aglycosylated forms of the mouse IgG1 mAb (1B7-11), produced in vitro in the presence and absence of tunicamycin. Charge heterogeneity was shown not to be a consequence of glycosylation status. Intracellular and secreted IgG mAbs were also analyzed to investigate the time course of change in charge properties of the heavy and light chains. The charge heterogeneity was found to be generated intracellularly, and alterations in charge properties could be induced during incubation under physiological conditions. Semilogarithmic plots of the density of the principal heavy and light chain spots against incubation time showed linear relationships, suggesting that the charge shifts result from a first-order reaction. The semilogarithmic plot for the light chain correlated well with the time after IgG synthesis. These results suggest that the charge heterogeneity of an IgG mAb is due to intra- and extracellular modifications of the polypeptide chains which reflect “aging” of antibody molecules.
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  • 46
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    Electrophoresis 19 (1998), S. 782-787 
    ISSN: 0173-0835
    Keywords: Two-dimensional zymography ; Serine proteinase ; Metalloproteinase ; Pure pancreatic juice ; Pancreatic cancer ; Proteinase inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Proteolytic enzymes in human pure pancreatic juice (PPJ), which was collected by cannulating the main pancreatic duct using endoscopy, were investigated by two-dimensional zymography (2-DZ). 2-DZ was carried out by combining isoelectric focusing (IEF) in the first dimension with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, using gels containing casein or gelatin as a substrate for the proteolytic enzymes. After electrophoresis, the gels were incubated in Triton X-100 followed by incubation at 37°C in Tris buffer (pH 8.5) containing CaCl2. By staining the gels with Coomassie Brilliant Blue (CBB R-250), proteolytic enzymes were detected as clear spots and zones against a blue background. Proteinase inhibitors, such as a cysteine proteinase inhibitor (E-64), a metalloproteinase inhibitor (EDTA), and a serine proteinase inhibitor (Pefabloc® SC), were added to PPJ in order to determine the types of proteinases. In patients with pancreatic cancer, spots of molecular weight (Mr) 70 000 and isoelectric points (pI) 5.3-5.5 were clearly detected on the gels containing casein and gelatin, while these spots were not detected in the PPJ from healthy subjects. The proteolytic activities of these spots were strongly inhibited by EDTA and Pefabloc SC but not E-64. These results suggest that the spots of Mr 70 000 and pI 5.3-5.5 in PPJ of pancreatic cancer might be matrix metalloproteinase 2, which is a candidate for tumor-associated proteinase. 2-DZ proved to be a tool for analysis of proteolytic enzymes in PPJ and for the clinical diagnosis of pancreatic cancer.
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  • 47
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    Electrophoresis 19 (1998), S. 818-825 
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Human breast carcinoma proteins ; Tandem mass spectrometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Previously, we reported a two-dimensional gel map and database with molecular weight/isoelectric point (Mr/pI) loci for 22 proteins expressed in the breast carcinoma cell line, MDA-MB231 (Rasmussen et al., Electrophoresis 1997, 18, 588-598). Here we update this database with Mr/pI loci for a further nine cytoplasmic proteins and three Triton X-114 solubilised membrane proteins from MDA-MB231 cells. In addition, a novel protein, previously represented only in expressed sequence tag (EST) databases, has been identified as a Triton X-114 soluble protein and assigned an Mr/pI locus. During the course of isolating proteins from the Triton X-114 fraction, we compared recoveries of proteins in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels after isoelectric focusing (IEF) using either immobilised pH gradients or carrier ampholytes. In these experiments, a significantly higher proportion of membrane proteins were visible in SDS-polyacrylamide gels after the use of carrier ampholytes for the first dimension. We also report our mass spectrometric-based procedure for identifying two-dimensional electrophoresis (2-DE) gel-resolved proteins, combining in-gel enzymatic digestion, 0.2 mm internal diameter (ID) capillary column reversed-phase high-performance liquid chromatography (RP-HPLC) peptide mapping and electrospray ionisation  -  ion trap  -  mass spectrometry.
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  • 48
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    Electrophoresis 19 (1998), S. 2506-2514 
    ISSN: 0173-0835
    Keywords: Affinity electrophoresis ; Titration curves ; Albumin ; Animal species ; Cibacron Blue ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Albumins of different species, varying in electrophoretic mobility, were compared in their interaction with the dye Cibacron Blue F3G A. Immobilized by coupling to a high molecular weight dextran (“blue dextran”), the dye was used as a ligand in affinity electrophoresis in different setups. One-dimensional electrophoresis with blue dextran entrapped in an intermediate gel and two-dimensional applications with transverse gradients (affinity titration curves, zonal electrophoresis in linear ligand gradients) were performed. Compared to the human homologue, animal albumins display more complex patterns and interaction profiles, depending on pH and ionic strength of the buffers. Results may differ considerably from those obtained by affinity chromatography, illustrating the additional screening potential of the electrophoretic methods. Comparison of different samples under the influence of ligand competition, reducing conditions, or denaturing agents gives supplementary information on conformational behavior of the proteins.
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  • 49
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    Electrophoresis 19 (1998), S. 2528-2535 
    ISSN: 0173-0835
    Keywords: Small GTPases ; Phagosome ; Two-dimensional polyacryl-amide gel electrophoresis ; Tetrahymena thermophila ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In this study we purified phagosomes of the ciliated protozoan Tetrahymena thermophila to analyze aspects of the maturation pathway of phagocytotic vesicles. Phagosomes were labeled with magnetic microparticles and then purified in high amounts with the help of a permanent magnet. By combining a pulse-chase labeling protocol with the magnetic separation procedure we were able to isolate phagosomes of defined ages, which represent distinct stages of their maturation pathway. GTP-overlay assays showed that a set of small GTPases of the ras superfamily is associated with these phagosomes. Phagosomes isolated at different stages of maturation revealed a change in the pattern of the small GTPases. Some small GTPases identified by the GTPase overlay assays could be aligned to India ink stained protein spots in two-dimensional gels of isolated phagosomes. The results presented here are a first step to identify the members of small GTPases associated with phagosomes during their maturation pathway. Microsequencing of pooled polypeptides by mass-spectrometric techniques will identify the primary structure of these small GTPases.
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    Electrophoresis 19 (1998), S. 2536-2536 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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    Electrophoresis 19 (1998) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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    Electrophoresis 19 (1998), S. i 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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    Electrophoresis 19 (1998), S. 2539-2560 
    ISSN: 0173-0835
    Keywords: Capillary electrophoresis ; Simple carbohydrates ; Glycoprotein glycans ; Glycopeptides ; Glycoforms ; Glycolipids ; Glycosaminoglycans ; Review ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: This review summarizes publications on capillary electrophoresis (CE) of carbohydrates, covering almost all hitherto published papers on this topic. It is designed to be a convenient tool for the literature search by providing a comprehensive table. Since CE analysis of carbohydrates is generally complicated due to the structural diversity of carbohydrate species, an attempt is made in this table to supply detailed information on the analyzed form (underivatized or derivatized, type of derivative) and analytical conditions (capillary size, state of the inner wall, composition of the electrophoretic solution, applied voltage, detection method, etc.), for each combination of carbohydrate species to be analyzed. In addition, a brief overview is presented to help in the literature search.
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  • 54
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    Electrophoresis 19 (1998), S. 2595-2602 
    ISSN: 0173-0835
    Keywords: Lectin affinity electrophoresis ; Glycoforms ; α-Fetoprotein ; Complex-type oligosaccharide ; Glycoprotein ; Review ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: α-Fetoprotein (AFP) glycoforms, defined as AFP with different chemical structures of carbohydrate, were analyzed by affinity electrophoresis with several lectins of known specificities against complex-type oligosaccharides. Serum AFP samples from cord blood on full-term delivery and from patients with hepatocellular carcinoma and extrahepatic malignancies including gastrointestinal tumors and yolk sac tumors were used. Two-dimensional lectin affinity electrophoresis and also lectin affinity chromatography coupled with lectin affinity electrophoresis were employed. More than ten AFP glycoforms were identified or characterized using the above-mentioned AFP samples. Known specificities of the lectins against complex-type oligosaccharides were refined or their additional specificities were found in this study. Lectin appeared to have specificity against carbohydrates by recognizing not only specific residues but also the whole carbohydrate molecule containing the residues, resulting in differential affinities for the lectin.
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    Electrophoresis 19 (1998), S. 2603-2611 
    ISSN: 0173-0835
    Keywords: Starch ; Capillary electrophoresis ; Oligosaccharide ; Fluorophore-assisted carbohydrate electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The analysis of the fine structure of starches is important to the investigation of linkages between starch structure and function and to the investigation of the properties and roles of starch biosynthetic, modifying and degradation enzymes. Fluorophore-assisted carbohydrate electrophoresis has recently been introduced as a method for the analysis of the oligosaccharide populations released by the enzymatic digestion of starches, which has advantages in resolution and sensitivity over previously used methods, and provides the capacity for the facile analysis of oligosaccharide populations on either a molar or mass basis. The use of fluorophore-assisted carbohydrate electrophoresis for the analysis of oligosaccharides is reviewed with particular reference to the choice of label, efficiency of labeling and separation techniques. Examples of separations using slab gel electrophoresis, DNA sequencer analysis and capillary electrophoresis are presented and we conclude that on the basis of resolution and reproducibility, capillary electrophoresis is the method of choice for the separation of oligosaccharides of degree of polymerization from 1 to 100. Examples of isoamylase-debranched starches and glycogens analyzed by capillary electrophoresis are presented. The capillary electrophoresis analysis of starch structure through the analysis of oligosaccharides released by the debranching of limit dextrins derived from starches and glycogens is introduced as a useful diagnostic of starch structure. The potential for future development of novel diagnostics for starch structure using fluorophore-assisted carbohydrate electrophoresis is discussed.
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  • 56
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    Electrophoresis 19 (1998), S. 837-844 
    ISSN: 0173-0835
    Keywords: Membrane proteins ; Solubility ; Two-dimensional polyacrylamide gel electrophoresis ; Proteome ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.
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    Electrophoresis 19 (1998), S. 877-877 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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    Electrophoresis 19 (1998), S. 877-877 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    Electrophoresis 19 (1998), S. 878-878 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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    Electrophoresis 19 (1998), S. 860-866 
    ISSN: 0173-0835
    Keywords: Bacterial protein expression vector ; Recombinant proteins ; Polymerase chain reaction ; Protein purification ; Biotin affinity chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Expression of recombinant proteins is an important method for the characterisation of the structure and function of proteins. However, many expression methods can be difficult, time-consuming and lead to low protein yields. The Promega Pinpoint Xa1-T vector system is a unique, one-step cloning method that allows the direct insertion of polymerase chain reaction (PCR) fragments into the expression vector. We describe our experience of the use of this system to clone and express three proteins (8-12 kDa) directly from their PCR products. The proteins are expressed as fusion proteins with a 13 kDa biotinylated tag that can be used for detection of the expressed protein and affinity purification. In our case, the yield was greater than 20 mg per litre of culture. Expressed proteins were purified by Q-Sepharose anion-exchange chromatography and reverse-phase high-performance liquid chromatography (HPLC) instead of the conventional method of avidin-biotin affinity chromatography. The Pinpoint vector proved to be a relatively simple and fast protein expression technique suitable for wide application for expressing recombinant proteins.
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    Electrophoresis 19 (1998) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000