Elsevier

Analytical Biochemistry

Volume 437, Issue 1, 1 June 2013, Pages 104-108
Analytical Biochemistry

Recorded scan times can limit the accuracy of sedimentation coefficients in analytical ultracentrifugation

https://doi.org/10.1016/j.ab.2013.02.011Get rights and content

Abstract

We report systematic and large inaccuracies in the recorded elapsed time in data files from the analytical ultracentrifuge, leading to overestimates of the sedimentation coefficients of up to 10%. This far exceeds previously considered factors contributing to the uncertainty in this parameter and has significant ramifications for derived parameters such as hydrodynamic shape and molar mass estimates. The source of this error is currently unknown, but we found it to be quantitatively consistent across different instruments, increasing with rotor speed. Furthermore, its occurrence appears to correlate with the use of the latest data acquisition software from the manufacturer, in use in some of our laboratories for nearly 2 years. Many of the recently published sedimentation coefficients may need to be reexamined. The problem can be easily recognized by comparing the file timestamps provided by the operating system with the elapsed scan times recorded within the data files. Therefore, we implemented a routine in SEDFIT that can automatically examine the data files, alert the user to significant discrepancies, and correct the scan times accordingly. This eliminates errors in the recorded scan times.

Section snippets

Materials and methods

Sedimentation velocity (SV) experiments were carried out on Beckman Coulter analytical ultracentrifuges (Indianapolis, IN, USA) running the manufacturer’s data acquisition software ProteomeLab XL-I Graphical User Interface version 5.8 (firmware 4.21) in instrument A and ProteomeLab XL-A/XL-I Graphical User Interface version 6.0 (0419111340) (firmware 5.06) in instrument B. Samples of bovine serum albumin (BSA) were prepared by reconstituting lyophilized powder (cat. No. 7030, Sigma–Aldrich, St.

Results

Over the past 2 years, we have carried out an extensive comparison of SV data among a group of approximately 11 instruments in our joint laboratories. As will be described elsewhere in detail (Ghirlando et al., manuscript in preparation), we have found that BSA is a suitable and stable standard for SV. A typical SV data set is shown in Fig. 1, from which it can be discerned that a c(s) sedimentation coefficient distribution analysis provides excellent fits. The BSA monomer species is baseline

Discussion

The unexpected and systematic error we discovered in the reported passage of time from the start of centrifugation causes a systematic overestimate of sedimentation coefficients typically by approximately 10% at commonly used high rotor speeds. It appears to be associated with the manufacturer’s data acquisition software version 6.0 and/or related firmware, which was installed on the affected instruments during the spring and summer of 2011. We believe that it is installed in a significant

Acknowledgments

This work was supported by the Intramural Research Programs of the National Institute of Biomedical Imaging and Bioengineering, National Institute of Diabetes and Digestive and Kidney Diseases, and National Heart, Lung, and Blood Institute, National Institutes of Health.

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