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  • 1
    Electronic Resource
    Electronic Resource
    Computer simulation of the directional displacement of rod-shaped, arc-shaped, and circular objects in an array of obstacles, representing a simple model for the gel electrophoresis of small DNA (1995)
    New York : Wiley-Blackwell
    Biopolymers 35 (1995), S. 179-185 
    Details
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The gel electrophoresis of DNA of identical length but various static conformations was simulated using a two-dimensional model of the movement of rod-shaped, arc-shaped, and circular objects through random arrays of disk-shaped obstacles. At low obstacle density, the displacement rate of these objects decreases from the rod-shaped to the circular to the arc-shaped objects. At high obstacle densities, the displacement rate of circular objects approaches zero. The alignment of the arc-shaped objects along the axis of the directional movement of the objects was less than that achieved by the rod-shaped objects. Rod-shaped and arc-shaped objects were retarded in their movement by collisions with the obstacles; the number of collisions of the former, in view of their greater ability to align, was less than that of the latter. Circular objects were exclusively retarded by collisions, while the arc-shaped objects exhibited an additional retarding mechanism, viz. the suspension (“hanging”) on the obstacles. When the rigid objects were made flexible, their displacement increased. The increase was most pronounced with the circular objects, allowing them to penetrate at obstacle densities from which the rigid objects were excluded. © 1995 John Wiley & Sons, Inc.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bip.360350206
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  • 2
    Electronic Resource
    Electronic Resource
    Electrophoresis of proteins in semidilute polyethylene glycol solutions: Mechanism of retardation (1997)
    New York : Wiley-Blackwell
    Biopolymers 42 (1997), S. 183-189 
    Details
    ISSN: 0006-3525
    Keywords: electrophoresis ; retardation ; native proteins ; semidilute polymer solution ; scaling theory ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The retardation of proteins in the Mt range of 15-500 kDa in capillary electrophoresis conducted in semidilute solutions of the polymer polyethylene glycol (Mt range 0.2-8.0 × 106), was measured. The purpose was to test the predictions of the scaling theory with regard to the relation of retardation to (a) the Mt of the polymer, (b) the concentration of the polymer, and (c) the radius of the protein particles. These predictions derive from a mechanism that relates retardation to the screening length of the polymer solution, viewed as the average distance between the entanglement points of polymer chains.For the molecular weight range from 60 to 500 kDa of (near) spherical proteins, the retardation was found to be related to polymer concentration c asμ/μo = exp(-Ac0.69)where μ/μ0 is the retardation expressed as the ratio between the mobility in polymer solution and that in free solution. The value of the exponent of 0.69 is in close agreement with the value of 0.75 predicted by the scaling theory. Parameter A was found (a) to scale as the 0.04th power of Mt (polymer), approximating the predicted value of 0; and (b) to be proportional to particle radius as predicted. All measured values of retardation were independent of electric field strength in the range of 37-370 V/cm. Thus, experimental findings are consistent with the mechanism relating electrophoretic retardation to the screening length of the polymer network in the specified molecular weight range of proteins.Under the same conditions, log(μ/μ0) of proteins with Mt's less than 60 kDa (a) scales as the -0.06th power of Mt (polymer), and (b) is proportional to polymer concentration, suggesting a retardation mechanism that is not related to the screening length. © 1997 John Wiley & Sons, Inc. Biopoly 42: 183-189, 1997
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    The relative contributions of dispersion and diffusion to band spreading (resolution) in gel electrophoresis (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 1416-1419 
    Details
    ISSN: 0173-0835
    Keywords: Band spreading ; Resolution ; Diffusion ; Dispersion ; Agarose gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: DNA of approximately 2 kbp in length was previously found not to diffuse significantly in 1-1.5% agarose gels in the absence of an electric field, but to disperse during electrophoresis (Yarmola, E., Chrambach, A., Electrophoresis 1995, 16, 345-349). Accordingly, a process distinct from diffusion, and responsible for band spreading with migration time in gel electrophoresis, was defined as dispersion. Correspondingly, the diffusion coefficient, D(diff), was distinguished from a dispersion coefficient, D(disp). For DNA of approximately 1, 2 and 3 kbp, D(diff) and D(disp) were measured in agarose gel electrophoresis (1.0% SeaKem GTG). In that order of DNA length, D(disp) / D(diff) was found to increase from 5 to 15 to 45, showing that with increasing DNA length, time-dependent band spreading, and thus resolution in gel electrophoresis, is governed predominantly by dispersion, not diffusion. It is assumed that the essential part of electrophoretic dispersion is due to entanglement of the DNA molecule in the gel. Indirect evidence for such an entanglement derives from the observation of peak asymmetry and its interpretation by the Giddings-Weiss model.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170904
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  • 4
    Electronic Resource
    Electronic Resource
    Enhanced field strength and resolution in gel electrophoresis upon substitution of buffer by histidine at its isoelectric point (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 699-703 
    Details
    ISSN: 0173-0835
    Keywords: Electrophoresis ; Isoelectric histidine ; Field strength ; Dispersion coefficient ; Resolution ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Gel electrophoresis in isoelectric buffers, recently introduced by R. Westermeier and H. Schickle (Electrophoresis '95, Paris, Abstract No. 3, 1995), was applied to the automated HPGE-1000 apparatus in the expectation to be able to increase the field strength under the limiting conditions of heat dissipation capacity and voltage of that apparatus. A previous attempt to achieve that aim by reduction of gel thickness had not yielded more than a twofold increment in resolving power. Replacing 0.2 × Tris-boric acid-EDTA (TBE) buffer, conventionally applied in the apparatus at 15 V/cm, by 0.05 M histidine, pH 7.6 (close to the pI of 7.47), allows one to increase the field strength to 60 V/cm, thus providing a nearly fivefold increment in resolution under otherwise identical conditions (fluorescein carboxylate-labeled conalbumin-sodium dodecyl sulfate (SDS) and soybean trypsin inhibitor-SDS samples, 10°C, 4% MetaPhor agarose). An additional decrease in band dispersion can be obtained by decreasing the starting zone width through buffer dilution in the sample phase.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170413
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  • 5
    Electronic Resource
    Electronic Resource
    Interpretation of electrophoretic band shapes by a partition chromatographic model (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 1325-1332 
    Details
    ISSN: 0173-0835
    Keywords: Peak asymmetry ; Gaussian peak shape ; Non-Markovian diffusion ; Conalbumin ; Phycoerythrin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Measurements of the shape of electrophoretic bands of phycoerythrin and conalbumin have been made at regular intervals during migration in agarose gels. Analysis of the peak shapes suggests the existence of a significant degree of asymmetry. This is to be contrasted with the symmetry around the peak associated with the generally assumed Gaussian band. The degree of asymmetry of the bands decreased as a function of time and increased with agarose concentration. A similar experiment on DNA indicated constancy of the degree of asymmetry as a function of time. These results can be interpreted as, but do not prove the validity of, a nonlocal diffusion equation which generalizes a theory originally put forth by Giddings and Eyring (J. Am. Chem. Soc. 1955, 59, 416-420). The results may be significant in framing a measure of the resolvability of electrophoretic peaks.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170809
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  • 6
    Electronic Resource
    Electronic Resource
    Separation of viable from radiation-induced apoptotic lymphocytes by free-flow electrophoresis (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 1211-1214 
    Details
    ISSN: 0173-0835
    Keywords: Apoptotic lymphocytes ; Isolation ; Free-flow electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A human lymphocyte population undergoing apoptosis in vitro due to γ-irradiation was fractionated by free-flow electrophoresis in triethanolamine - Na-acetate buffers, containing up to 50 mM NaCl, with pH 6.0, 7.2 and 8.5, made isotonic by addition of sucrose. As shown by a flow cytometric analysis of the eluate, the distribution of apoptotic lymphocytes is shifted to the range of higher electrophoretic mobilities relative to that of viable ones at pH 8.5, yielding cell fractions enriched in apoptotic cells by a factor of 3 to 5. The difference in rates of electrophoretic migration observed at a mildly alkaline pH but not at a neutral or mildly acidic one suggests that the surface of apoptotic lymphocytes is more acidic than that of viable ones.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190723
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  • 7
    Electronic Resource
    Electronic Resource
    The band areas of proteins determined by fluorescent scanning in the commercial automated gel electrophoresis apparatus (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 1625-1630 
    Details
    ISSN: 0173-0835
    Keywords: Band area ; Protein ; Automated electrophoresis ; Automated apparatus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: An automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)-proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self-quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near-constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150191018
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  • 8
    Electronic Resource
    Electronic Resource
    Towards predicting mobility and resolution in polymeric media: Some first steps (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 1284-1287 
    Details
    ISSN: 0173-0835
    Keywords: Electrophoresis ; Polymer media ; Mobility ; Resolution ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Particle size-dependent retardation (“molecular sieving”) in electrophoresis can be achieved in polymer solutions or gels. In the semidilute concentration range of polymer solutions, mobility of “rigid, spherical” particles can be predicted from their size (in the size range less than 20 nm radius), the screening length specific for the particular polymer and a constant that can be experimentally determined for a polymer. That constant could be universal for all hydrophilic uncharged polymers. Band spreading in polymer solutions is constant over a range of particle sizes and polymer concentrations and increases beyond that range. Presumably, the critical division between the two ranges occurs when the diameter of the particle exceeds the screening length of the polymer network. Resolution, defined as separation divided by the sum of bandwidths, can thus be predicted for a limited particle size and polymer concentration range. In gels, resolution can be estimated from the slopes and free mobility intercepts of the Ferguson plot. However, the previous computation of resolution needs to be revised in view of the fact that band spreading in gels proceeds in proportion to time, presumably through interaction with the polymer, and not as a function of the square root of time as would be the case if band spreading resulted from diffusion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190811
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  • 9
    Electronic Resource
    Electronic Resource
    Commercial automated gel electrophoresis apparatus: Application to DNA, band dispersion, nonlinear Ferguson curves, and isolation (1995)
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 713-718 
    Details
    ISSN: 0173-0835
    Keywords: Gel electrophoresis ; Automation ; Dispersion ; Preparativeness ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Recently available commercial automated gel electrophoresis apparatus with intermittent scanning of fluorescently labeled gel patterns (the HPGE-1000 apparatus of LabIntelligence, Menlo Park CA) was tested with regard to (i) its applicability to DNA in its native conformation, (ii) its ability to recognize the correct number of components, (iii) its capability to evaluate the width and shape of bands detected during electrophoresis, (iv) its ability to yield nonlinear Ferguson plots in a labor-saving fashion, and (v) its preparative potential. Ethidium homodimer (EtD) DNA (bp) ratios were systematically varied and the mobility of DNA fragments labeled at each ratio was measured in order to find a ratio which provided an unaltered mobility and presumably therefore an unaltered conformation of the fragment. That ratio was found to be 1/40 EtD/ DNA (bp) or less. With such weak labeling of DNA, a representative fragment of 527 bp length requires a minimum load of 200 ng and a 2 μg load for a full-scale peak height. Using the baseline automatically selected by the software of the apparatus, the band areas of the 17 components of a DNA digest were consistently evaluated by the software, as evidenced by the proportionality between DNA length and area. The areas of the separated bands of DNA fragments of 1857 and 121 bp length were found to be constant with time of electrophoresis. The dispersion coefficient was found to decrease with agarose concentration in electrophoresis at 1 V/cm; however, at higher field strength, the band width of the 1857 bp fragment was surprisingly found to increase with gel concentration, presumably due to stretching. Electrophoretic band dispersion was not found to be due to diffusion since zone spreading in the absence of the electric field under otherwise identical conditions does not proceed measurably. For the discontinuous buffer system of Wiltfang et al. (Electrophoresis 1993, 12, 352-366) the resolving power, in terms of theoretical plate equivalents, was approximately threefold higher compared to that in the Trisborate-EDTA (TBE) buffer under otherwise identical conditions. Two electrophoretic runs of the apparatus with different gel concentrations in its eight channels suffice to define nonlinear Ferguson curves, provided that the free mobility is measured by capillary electrophoresis. The commercial automated apparatus excels as a preparative device by allowing one to monitor the qualitative and quantitative success of electroelution.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501601115
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  • 10
    Electronic Resource
    Electronic Resource
    Dispersion coefficients of a protein and DNA fragment in polyacrylamide gel electrophoresis as a function of parameters defining the effective gel pore size and particle size (1995)
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 895-898 
    Details
    ISSN: 0173-0835
    Keywords: Band dispersion ; Polyacrylamide gel ; Uncrosslinked polyacrylamide solutions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The dispersion coefficient, D′, of the representative homogeneous protein, conalbumin, decreases linearly as the polyacrylamide concentration increases from 4 to 14%T (2%C), and varies in a biphasic fashion as %C (Bis) is increased from 2 to 20%, with a broad peak between 5 and 15%C. D′ increases linearly with the concentration of the initiator, potassium persulfate, in the range of 0.01-0.15%. D′ remains constant when the field strength is varied from 5 to 15 V/cm. A DNA fragment (1857 bp) exhibits a constant D′ in 4-6% polyacrylamide (2%C) at a field strength of 1 V/cm, and a linearly increasing D′ at 5 V/cm, in analogy to its previously observed behavior in agarose gels. In solutions of uncrosslinked polyacrylamide, the decrease of the D′ of conalbumin with polymer concentration is not significantly different from that in 2% N,N′-methylenebisacrylamide-crosslinked gels in the range of 4-14%T, while the decrease of mobility with polyacrylamide concentration is much steeper in 2% crosslinked compared to uncrosslinked polymer. Finally, -∂ (log D′)/∂T was found to be proportional to the retardation coefficient, KR (= -∂ (log μ)/∂T), in polyacrylamide gels. The ratio of -∂ (log D′)/∂T over KR increases with field strength in the range of 5-15 V/cm.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501601150
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