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By Cole, James L

Analytical Ultracentrifugation, the most recent quantity in Methods in Enzymology, makes a speciality of analytical ultracentrifugation. The scope of this method has vastly extended lately as a result of advances in instrumentation, algorithms and software program.

This quantity describes the newest ideas within the box and within the functions of analytical ultracentrifugation within the research of macromolecules, macromolecular assemblies, and biopharmaceuticals.

  • Timely contribution that describes a speedily altering field
  • Leading researchers within the field
  • Broad insurance: instrumentation, easy conception, facts research, and applications

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A fiber option that does not exhibit this effect is being investigated. Nevertheless, by correcting the data with a conversion to pseudo-absorbance Fig. 4, the quality was sufficient for a thorough evaluation of the species present in the sample. Solarization and the intensity instability phenomena are observed to be more pronounced with higher lamp power, but are almost completely eliminated when light below 300 nm is blocked by a high-pass filter. Therefore to maximize fiber life, the flash lamp power is set as low as possible, and UV light is filtered out when only measuring in the visible.

013)). Inclusion of greatly varying concentrations assures that the dynamic range of the spectrophotometer can be optimally used for even greatly varying extinction ranges at different wavelengths. Our implementation in UltraScan allows the user to exclude data above a threshold optical density to assure that only linear data are included in the fit. A benefit of the global fitting of multiple concentrations at multiple wavelengths is the ability to span greater concentration ranges, because extinction coefficients vary considerably over a large wavelength range for most biological macromolecules, especially in the low UV range below 230 nm.

APPLICATIONS To illustrate the capabilities of the MWL instrument and to compare MWL performance to traditional single-wavelength AUC, we characterized mixtures containing different ratios of DNA and protein (20:80, 35:65, 50:50, 65:35, and 80:20 on a per volume basis) and determined both the hydrodynamic properties of all components and identified the ratios of DNA and protein in each mixture from the AUC analysis. 013)). 1. Hydrodynamic characterization by traditional AUC: After validating the composition via spectral decomposition, the same samples were measured by AUC in the Beckman-Coulter XL-A at 258 and 278 nm, at 20 °C, 28,000 rpm using epon-charcoal 2-channel centerpieces (Beckman-Coulter) to verify the protein:DNA ratios with an independent method.

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