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  • 1
    Electronic Resource
    Electronic Resource
    Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels: Generalization of the reverse-staining technique (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 2398-2406 
    Details
    ISSN: 0173-0835
    Keywords: Detection ; Imidazole ; Negative staining ; Lysozyme ; Inflammation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We have recently shown that a few nanograms of protein separated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels can be detected by reverse-staining, exploiting the precipitation reaction between zinc(II) and imidazole. Modifications of this method have also been generated to detect gelisolated nucleic acids and bacterial glycolipids. Because there is no recourse to chemical modifiers, the reverse-staining technique has been valuable when micropreparing these biomacromolecules for later use or characterization. The mechanism underlying the reverse-staining effect, however, remains incompletely understood and this has prevented a further generalization of the technique. Here, we have conducted physicochemical experiments and identified zinc imidazolate (ZnIm2) as the main component of the precipitate that forms along the surface of zinc-imidazole reverse-stained gels. Many staining effects observed when gels containing electrophoretically separated biopolymers are subjected to zinc-imidazole stains have been rationalized. The reverse-staining method has been vastly generalized, now allowing the detection of proteins and glycolipids as well as complexes of these macromolecules in native gels. We demonstrate the application of the reverse-staining technique in situations where Coomassie blue or silver staining was inappropriate or failed to produce detection of the species of interest. The present generalization of the reverse-staining method facilitated the characterization of biomacromolecular interaction partners in mixtures of bacterial glycolipids and human tears.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150191407
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  • 2
    Electronic Resource
    Electronic Resource
    Zinc-imidazole positive: A new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 26-29 
    Details
    ISSN: 0173-0835
    Keywords: Zinc staining ; Gel electrophoresis ; Detection of nucleic acids ; DNA staining ; UV damage ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Nucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological a ctivity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV-light. As an alternative, for preparative purposes, a new non-toxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA, and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a few minutes, zinc-DNA complexes are visualized as deep-white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170105
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  • 3
    Electronic Resource
    Electronic Resource
    Single-step electrotransfer of reverse-stained proteins from sodium dodecyl sulfate-polyacrylamide gel onto reversed-phase minicartridge and subsequent desalting and elution with a conventional high-performance liquid chromatography gradient system for analysis (1995)
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 911-920 
    Details
    ISSN: 0173-0835
    Keywords: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ; Reverse staining ; Protein microanalysis ; High-performance liquid chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Isolation of proteins from polyacrylamide electrophoresis gels by a novel combination of techniques is described. A given protein band from a reverse stained (imidazol-sodium dodecyl sulfate - zinc salts) gel can be directly electrotransferred onto a reversed-phase chromatographic support, packed in a self-made minicartridge (2 mm in thickness, 8 mm in internal diameter, made of inert polymeric materials). The minicartridge is then connected to a high-performance liquid chromatography system and the electrotransferred protein eluted by applying an acetonitrile gradient. Proteins elute in a small volume (〉 700 μL) of high-purity volatile solvents (water, trifluoroacetic acid, acetonitrile) and are free of contaminants (gel contaminants, salts, etc). Electrotransferred proteins were efficiently retained, e.g., up to 90% for radioiodinated α-lactalbumin, by the octadecyl matrix, and their recovery on elution from the minicartridge was in the range typical for this type of chromatographic support, e.g., 73% for α-lactalbumin. The technique was successfully applied to a variety of proteins in the molecular mass range 6-68 kDa, and with amounts between 50 and 2000 pmol. The good mechanical and chemical stability of the developed minicartridges, during electrotransfer and chromatography, allowed their repeated use. This new technique permitted a single-step separation of two proteins unresolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to their different elution from the reversed-phase support. The isolated proteins were amenable to analysis by N-terminal sequencing, enzymic digestion and mass spectrometry of their proteolytic fragments. Chromatographic elution of proteins from the reversed-phase mini-cartridge was apparently independent of the specific loading mode employed, i.e., loading by conventional loop injection or by electrotransfer.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501601154
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  • 4
    Electronic Resource
    Electronic Resource
    A procedure for protein elution from reverse-stained polyacrylamide gels applicable at the low picomole level: An alternative route to the preparation of low abundance proteins for microanalysis (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 1564-1572 
    Details
    ISSN: 0173-0835
    Keywords: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ; Protein detection ; Protein micropurification ; Passive elution ; Trace enrichment ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We developed a technique that allows rapid protein elution from polyacrylamide gel bands at room temperature into a detergent-free buffer (elution time 2 × 10 min, total working time about 30 min) with high yields (90-98%) even at a low picomole level (1 picomole per band). Its efficacy relies on the combination of protein detection by reverse staining with the enhancement of protein diffusion after gel crushing. Detection is accomplished by gel incubation in an imidazole solution, followed by incubation in a zinc salt solution to develop a negative stain pattern. Proteins are eluted by zinc complexation in Laemmli electrophoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g. high performance liquid chromatography (HPLC) and automatic sequencing. A variety of proteins were eluted efficiently (with no apparent restriction due to their intrinsic properties) as quantified with radioiodinated total E. coli proteins. Yields were independent of acrylamide concentration, protein molecular mass (from 10 to 100 kDa) and the amount (from 1 to 100 picomole) of protein in the band. This protocol was derived from a quantitative evaluation of the effect of protein staining and of sample reduction prior to electrophoresis on elution yields. For N-terminal sequencing, the protein eluate was automatically loaded on a polyvinylidene difluoride (PVDF) membrane with conventional HPLC equipment; both loading and membrane clean-up were monitored at 206 nm. By simultaneously processing several analytical bands, the procedure allowed trace enrichment of a natural scarce protein that was N-terminal sequenced.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150171012
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