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  • 1
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 1 (1980), S. 72-72 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Ultrathin-layer isoelectric focusing in 50-100 μm polyacrylamide gels covalently bound to glass plates or polyester films, pretreated with methacryloxypropyl-trimethoxy-silane, is described. The gels adhere firmly to the silanized supports during all steps of operation and visualization. Ultrathin gels are prepared by the new and simple “flap technique”. Artificial mixtures of marker proteins and crude fungal enzymes were focused on 5-25 cm separation distances. In 11 cm gels containing pH 4-9 “Servalyt T” carrier ampholytes, 2-3 zones per mm can be resolved, with a zone width of 70-150 μm and differences in pI values of 0.023-0.033 pH. The gel volume per focused sample is 15-120 μl and up to 100 samples can be analyzed on 12.5 × 26 cm gels. Polyacrylamide gels of intermediate and high porosity, e. g. 5 % T, 3 % C and 3 % T, 20 % C, are compared. Small marker proteins attain equilibrium in both gels after focusing for 1300-2000 V x h at a final field strength of 100 V/cm, whereas thyroglobulin (690 000) and ferritin (465 000), applied at the anode and cathode, coalesce only in high-porosity gels. pH Gradients were determined, with the aid of a glass electrode, directly on the gel surface for wide range carrier ampholytes “Servalyt T”, “Ampholine”, and “Pharmalyte” after focusing for 2000-5000 V × h. Identical focusing patterns are found for some marker proteins and crude fungal enzymes in different carrier ampholytes. The effect of purification of commercial carrier ampholytes by ultrafiltration and charcoal treatment, and of “forced aging” at 80°C, on isoelectric focusing of marker proteins was studied. The molecular size distribution of different carrier ampholytes was assessed by thin-layer gel chromatography on Bio-Gel P-10. Protein staining with Serva Blue G, Serva Blue R and Serva Violet 49 is described. The sensitivity of protein detection is 50-100 ng/mm2. Fixation, staining and destaining can be completed within 10-15 min in 50 μm gels. Ultrathin-layer isoelectric focusing on silanized glass plates or polyester films recommends itself as an excellent analytical technique for research and routine applications. The method combines high resolution, speed, versatility and reagent economy with simplicity of operation.
    Additional Material: 14 Ill.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 1 (1980), S. 78-82 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: There are a number of methods-currently available for determining the molecular weights of RNA molecules by gel electrophoresis. This article reviews these techniques briefly and appraises the major advantages and disadvantages of each.
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  • 4
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A device for the measurement of segmental voltages along electrofocusing gels made of polyacrylamide has been designed and tested successfully. It allows for continuous monitoring of the changing voltage gradients which occur during electrofocusing. By means of 11 equidistant embedded platinum probes, the voltage drop at any one segment or sequential array of segments can be determined at any time without disturbing the ongoing electrofcusing. The wires are sealed into the wall of a 6 mm diameter (ID) Pyrex gel tube at 1.0 cm intervals. Measurements on a gel containing 2% Ampholine (pH-range 3.5-10) showed that the voltage gradient changes during electrofocusing. Starting with a uniform voltage drop of 10 ± 2%cm across a 10 cm gel, two transient peaks of high potential (comprising at 15 min 17% and 25% of the applied voltage) move progressively from the gel periphery toward its center. They coalesce into a broad single peak centered at 0.4 of the distance from the anodic gel terminus within 1.5 h. This major single peak remained stationary, changing slightly in magnitude during the next 18 h of electrofocusing. At this final stage, the voltage peak occurred at pH 6.3 and approximately 25% of the applied voltage was across this segment of gel.
    Additional Material: 3 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 1 (1980), S. 127-127 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 1 (1980), S. 129-136 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 1 (1980), S. 141-149 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A maximum utilization of the high resolving power of isoelectric focusing requires that the focused zones are both planar and perpendicular to the direction of focusing. This paper reports on extensive investigations carried out with a rectangular quartz column with provisions for direct photography of zonal shapes. The experiments have been performed in density gradients, but the conclusions may be generalized to other stabilizing media. It is demonstrated that distorted zones are formed if a second pH gradient, perpendicular to the direction of focusing, originates at one end of the column. The generation of such a pH gradient may have different causes: erroneous loading of the column, too high an initial power input, presence of salts of weak electrolytes in the sample, or unsuitable positioning of the electrodes. The effects of the second pH gradient on the shapes of the focused carrier-ampholyte and protein zones are interpreted on the basis of the mechanism creating the natural axial pH gradient. The stability of the disturbed system is discussed in terms of the diffusional, electrical, and convective mass flows of its components.
    Additional Material: 10 Ill.
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  • 8
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A natural pH gradient can be formed on polyacrylamide gel using either two (glutamic acid and lysine only) or three (glutamic acid, histidine and lysine) aminoacids as the sole carrier ampholyte species. In the two component system, glutamic acid and lysine focus as single peaks adjacent (anodically and cathodically, respectively) to the steep, linear center of the step-function pH gradient. Histidine, in the three component system, focuses at the center of the pH gradient and separates the other two aminoacids. Each aminoacid electrofocuses at its pI. Hemoglobin electrofocuses as a sharp band in both systems and, in the two component system, remains focused at its pI for 200 h. A major conductance gap forms at the gel center in both systems with progressing time of electrofocusing; a minor and transient conductance gap develops in the glass tube just above the gel.
    Additional Material: 4 Ill.
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  • 9
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Methods of two-dimensional protein fractionation have been analysed for their suitability for the analysis of heterogeneous ribonucleoprotein (hnRNP) particles. Nonequilibrium pH gradient electrophoresis gives the best overall fractionation and reveals that the dominant “core” proteins possess charge heterogeneity. For the analysis of the minor components of the particles, it may be preferable to overload isoelectric focusing gels and allow the basic core proteins, which comprise 70% of the total protein, to migrate off the gel. This does, however, result in the loss of some basic minor polypeptides.
    Additional Material: 4 Ill.
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  • 10
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The present paper gives a description of a procedure for isolation of pepsinogen I from human urine. The initial steps are concentration by batch adsorption to DEAE-Sephadex, desorption by addition of NaCl, and DEAE-chromatography. The final step is preparative isotachophoresis with Ampholine carrier ampholytes as spacers. Exact visualization of the fractionations is obtained by fused rocket immunoelectrophoresis. The procedure provides pepsinogen I of high purity as shown by crossed immunoelectrophoresis, agar gel electrophoresis and N-terminal aminoacid analysis. The yield of the procedure is 0.05-0.1 mg pepsinogen I per liter urine.
    Additional Material: 5 Ill.
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