A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation
Date
2015-05-21
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PLOS (Public Library of Science)
Abstract
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute
sedimentation coefficients and buoyant molar masses of macromolecules and their complexes,
reporting on their size and shape in free solution. The purpose of this multi-laboratory
study was to establish the precision and accuracy of basic data dimensions in AUC and
validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing
radial and temperature calibration tools and a bovine serum albumin (BSA) reference
sample were shared among 67 laboratories, generating 129 comprehensive data
sets. These allowed for an assessment of many parameters of instrument performance, including
accuracy of the reported scan time after the start of centrifugation, the accuracy of
the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation
coefficients obtained for BSA monomer in different instruments and using different
optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of
(4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from
the external calibration references for elapsed time, scan velocity, temperature, and radial
magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold
reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an
opportunity to determine the instrument-to-instrument variation of the absolute radial positions
reported in the scan files, the precision of photometric or refractometric signal magnitudes,
and the precision of the calculated apparent molar mass of BSA monomer and the
fraction of BSA dimers. These results highlight the necessity and effectiveness of independent
calibration of basic AUC data dimensions for reliable quantitative studies.
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Zhao H, Ghirlando R, Alfonso C, Arisaka F, Attali I, Bain DL, et al. (2015) A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation. PLoS ONE 10(5): e0126420. doi:10.1371/journal.pone.0126420