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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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VCU Physiology and Biophysics Publications
Long-term stability of iButton temperature readings.
S1_Table.pdf (55 kB)
Data underlying the statistical analyses.
S2_Fig.tif (327 kB)
Long-term stability of the radial mask.
S3_Fig.tif (216 kB)
Long-term stability of the cell assembly.
S4_Fig.tif (201 kB)
Long-term trend in the monomer signal.
S5_Fig.tif (194 kB)
Long-term trend in the dimer fraction.
S6_Fig.tif (162 kB)
Indicators of convection as a function of initial temperature jump.
S7_Fig.tif (554 kB)
Example for a data set with likely initial convection.
S8_Fig.tif (602 kB)
Example for a data set with sloping plateaus.
S9_Fig.tif (177 kB)
Correlation between monomer and dimer signal.
S10_Fig.tif (126 kB)
Absence of correlation between monomer signal and nominal acquisition wavelength.
S11_Fig.tif (150 kB)
Correlation between absorbance and interference signals.