Ultrathin-layer sodium dodecyl sulfate gel electrophoresis of proteins: Effects of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes

Gerstner, Árpád; Csapó, Zsolt; Sasvári-Székely, Mária; Guttman, András

doi:10.1002/(sici)1522-2683(20000301)21:5<834::aid-elps834>3.0.co;2-x

Cited by 16 publications

(12 citation statements)

References 32 publications

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“…4B depicts, E a values for electrophoresis microchip separation of dsDNA fragments using PVP solution exhibited a slightly elevating tendency with increasing solute size (23±23.25 kJ/mol), suggesting that electric field mediated migration of the larger dsDNA fragments require a somewhat higher activation energy to warp through the dynamically structured polymer network. Similar increasing plots were observed earlier in CGE of ssDNA sequencing fragments in cross-linked polyacrylamide gels (19±24 kJ/mol) [34] and in UTLGE of similar size dsDNA fragments using composite hydroxyethyl cellulose-agarose (22±24 kJ/mol) and polyethylene oxide-agarose gels (18±20 kJ/mol) [35]. Figure 5 delineates the electrophoretic mobility values as the function of staining dye concentration ranging from 0.1 to 1 mM.…”

Section: Effect Of Separation Temperaturementioning

confidence: 99%

DNA analysis on electrophoretic microchips: Effect of operational variables

et al. 2001

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Applicability of modern microfabrication technology to electrophoresis microchips initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field mediated separations in microfabricated devices (electrophoresis microchips) are significantly faster than conventional gel electrophoresis, usually completed in seconds to minutes. Electrophoretic separation of DNA molecules on microfabricated devices proved to have the potential to improve the throughput of analysis by orders of magnitude. The flexibility of electrophoresis microchips allows the use of a plethora of separation matrices and conditions. In this paper, we report on electric field mediated separation of fluorescent intercalator-labeled dsDNA fragments in polyvinylpyrrolidone matrix-filled microchannel structures. The separations were detected in real time by a confocal, single-point laser-induced fluorescence/photomultiplier setup. Effects of the sieving matrix concentration (Ferguson plot), migration characteristics (reptation plot), separation temperature (Arrhenius plot), as well as applied electric field strength and intercalator concentration on the separation of DNA fragments are thoroughly discussed.

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Section: Effect Of Separation Temperaturementioning

confidence: 99%

DNA analysis on electrophoretic microchips: Effect of operational variables

et al. 2001

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“…Interestingly, the increasing dependence was noticed with cross-linked polyacrylamide [18], agarose [27] and composite HEC-agarose and PEO-agarose gels [28,29], while the decreasing dependence occurs with noncrosslinked polyacrylamide [19] or composite linear polyacrylamide-agarose gels [28,29]. Coming back to the results from this work, it is worth noting at this point that under 40 mM ionic strength the radius of gyration of the M r 177´10 3 PSS was estimated at 20 nm [16], which means that its diameter is very close to the blob size of the separating network.…”

Section: Influence Of the Sieving Polymer Concentration And Ionic Strmentioning

confidence: 74%

“…The effects of the chain length of the analyte, mesh size of the separating network, ionic strength of the electrolyte and electric field strength were investigated. A major issue of this study was to confront the results from this work with those obtained for nucleic acids and denatured proteins in similar polymer solutions [17±20, 22±25] and in other sieving matrices (chemical [21,26], physical [27,28] and composite gels [28,29]) investigated either in a capillary [21] or lately in the ultrathin layer format [26±29] (Table 1). To the best of our knowledge, no experimental data are available on the effect of temperature in the size-based separation of synthetic polymers.…”

Section: General Considerationsmentioning

confidence: 93%

On the use of the activation energy concept to investigate analyte and network deformations in entangled polymer solution capillary electrophoresis of synthetic polyelectrolytes

Cottet

Gareil

2001

Electrophoresis

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The activation energy associated with the electrophoretic migration of an analyte under given electrolyte conditions can be accessed through the determination of the analyte electrophoretic mobility at various temperatures. In the case of the electrophoretic separation of polyelectrolytes in the presence of an entangled polymer network, activation energy can be regarded as the energy needed by the analyte to overcome the obstacles created by the separating network. Any deformation undergone by the analyte or the network is expected to induce a decrease in the activation energy. In this work, the electrophoretic mobilities of poly(styrenesulfonates) (PSSs) of various molecular weights (Mr 16 x 10(3) to 990 x 10(3)) were determined in entangled polyethylene oxide (PEO) solutions as a function of temperature (in the 17-60 degrees C range) and the PSS activation energies were calculated. The influences of the PSS molecular weight, blob sizes zetab of the separating network (related to the PEO concentration), ionic strength of the electrolyte and electric field strength (75-600 V/cm) were investigated. The results were interpreted in terms of analyte and network deformations and were confronted with those previously obtained for DNA migration in polymer solutions and chemical gels. For a radius of gyration Rgzetab, suggesting PSS and network deformations in the latter case. Increasing ionic strength resulted in an increase in the PSS activation energy, because of the decrease of their radii of gyration, which makes them less deformable. Finally, the activation energies of all the PSSs are a decreasing function of field strength and at high field strength tend to reach a constant value close to that for a small molecule.

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“…It has been reported, from the thin layer electrophoresis experiment, that fields up to 89 V/cm does not affect the mobility of SDS-protein complexes [20]. Since our channel was much thinner (∼27 m) compared to the case of the thin-layer electrophoresis (∼180 m), it is less prone to gel heating problem.…”

Section: Resultsmentioning

confidence: 99%