Recorded scan times can limit the accuracy of sedimentation coefficients in analytical ultracentrifugation
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.
References (30)
- et al.
Editorial for the special issue of methods, “modern analytical ultracentrifugation”
Methods
(2011) - et al.
Analytical ultracentrifugation for the study of protein association and assembly
Curr. Opin. Chem. Biol.
(2006) - et al.
Measuring low levels of protein aggregation by sedimentation velocity
Methods
(2011) - et al.
Precise boundary element computation of protein transport properties: diffusion tensors, specific volume, and hydration
Biophys. J.
(2006) - et al.
Analytical ultracentrifugation combined with X-ray and neutron scattering: experiment and modelling
Methods
(2011) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling
Biophys. J.
(2000)- et al.
Rotavirus nonstructural protein NSP2 self-assembles into octamers that undergo ligand-induced conformational changes
J. Biol. Chem.
(2001) - et al.
Ribonucleoprotein particles from Escherichia coli
J. Mol. Biol.
(1959) - et al.
Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems
Biophys. J.
(2002) On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation
Anal. Biochem.
(2003)