Abstract
Experiments performed in the analytical ultracentrifuge (AUC) measure sedimentation and diffusion coefficients, as well as the partial concentration of colloidal mixtures of molecules in the solution phase. From this information, their abundance, size, molar mass, density and anisotropy can be determined. The accuracy with which these parameters can be determined depends in part on the accuracy of the radial position recordings and the boundary conditions used in the modeling of the AUC data. The AUC instrument can spin samples at speeds up to 60,000 rpm, generating forces approaching 300,000 g. Forces of this magnitude will stretch the titanium rotors used in the instrument, shifting the boundary conditions required to solve the flow equations used in the modeling of the AUC data. A second source of error is caused by the chromatic aberration resulting from imperfections in the UV–visible absorption optics. Both errors are larger than the optical resolution of currently available instrumentation. Here, we report software routines that correct these errors, aided by a new calibration disk which can be used in place of the counterbalance to provide a calibration reference for each experiment to verify proper operation of the AUC instrument. We describe laboratory methods and software routines in UltraScan that incorporate calibrations and corrections for the rotor stretch and chromatic aberration in order to support Good Manufacturing Practices for AUC data analysis.
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Acknowledgements
This work was supported by the Canadian Foundation for Innovation, the Canada 150 Research Chairs program, the Canada Foundation for Innovation Grant CFI-37589, National Institutes of Health grant 1R01GM120600, NSERC DG-RGPIN-2019-05637, a CIHR foundation grant (FDN 148469), and the Chinook Summer Studentship Award. Special thanks to Denton Fredrickson.
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Stoutjesdyk, M., Henrickson, A., Minors, G. et al. A calibration disk for the correction of radial errors from chromatic aberration and rotor stretch in the Optima AUC™ analytical ultracentrifuge. Eur Biophys J 49, 701–709 (2020). https://doi.org/10.1007/s00249-020-01434-z
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DOI: https://doi.org/10.1007/s00249-020-01434-z