A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Huaying Zhao, Rodolfo Ghirlando, Carlos Alfonso, Fumio Arisaka, Ilan Attali, David L. Bain, Marina M. Bakhtina, Donald F. Becker, Gregory J. Bedwell, Ahmet Bekdemir, Tabot M.D. Besong, Catherine Birck, Chad A. Brautigam, William Brennerman, Olwyn Byron, Agnieszka Bzowska, Jonathan B. Chaires, Catherine T. Chaton, Helmut Cölfen, Keith D. ConnaghanKimberly A. Crowley, Ute Curth, Tina Daviter, William L. Dean, Ana I. Díez, Christine Ebel, Debra M. Eckert, Leslie E. Eisele, Edward Eisenstein, Patrick England, Carlos Escalante, Jeffrey A. Fagan, Robert Fairman, Ron M. Finn, Wolfgang Fischle, José García De La Torre, Jayesh Gor, Henning Gustafsson, Damien Hall, Stephen E. Harding, José G. Hernández Cifre, Andrew B. Herr, Elizabeth E. Howell, Richard S. Isaac, Shu Chuan Jao, Davis Jose, Soon Jong Kim, Bashkim Kokona, Jack A. Kornblatt, Dalibor Kosek, Elena Krayukhina, Daniel Krzizike, Eric A. Kusznir, Hyewon Kwon, Adam Larson, Thomas M. Laue, Aline Le Roy, Andrew P. Leech, Hauke Lilie, Karolin Luger, Juan R. Luque-Ortega, Jia Ma, Carrie A. May, Ernest L. Maynard, Anna Modrak-Wojcik, Yee Foong Mok, Norbert Mücke, Luitgard Nagel-Steger, Geeta J. Narlikar, Masanori Noda, Amanda Nourse, Tomas Obsil, Chad K. Park, Jin Ku Park, Peter D. Pawelek, Erby E. Perdue, Stephen J. Perkins, Matthew A. Perugini, Craig L. Peterson, Martin G. Peverelli, Grzegorz Piszczek, Gali Prag, Peter E. Prevelige, Bertrand D.E. Raynal, Lenka Rezabkova, Klaus Richter, Alison E. Ringel, Rose Rosenberg, Arthur J. Rowe, Arne C. Rufer, David J. Scott, Javier G. Seravalli, Alexandra S. Solovyova, Renjie Song, David Staunton, Caitlin Stoddard, Katherine Stott, Holger M. Strauss, Werner W. Streicher, John P. Sumida, Sarah G. Swygert, Roman H. Szczepanowski, Ingrid Tessmer, Ronald T. Toth, Ashutosh Tripathy, Susumu Uchiyama, Stephan F.W. Uebel, Satoru Unzai, Anna Vitlin Gruber, Peter H. Von Hippel, Christine Wandrey, Szu Huan Wang, Steven E. Weitzel, Beata Wielgus-Kutrowska, Cynthia Wolberger, Martin Wolff, Edward Wright, Yu Sung Wu, Jacinta M. Wubben, Peter Schuck

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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.

Original languageEnglish
Article numbere0126420
JournalPLoS ONE
Issue number5
StatePublished - 21 May 2015


FundersFunder number
National Cancer InstituteCA35635
National Heart, Lung, and Blood Institute
National Institute of Biomedical Imaging and Bioengineering
National Institute of Diabetes and Digestive and Kidney Diseases
National Institute of General Medical SciencesGM 095822, P30GM103519, GM 109102, GM 094363
National Institutes of HealthGM067777, F31GM105363, GM82545, GM-29158
National Research Foundation of Korea2011-0010437
National Stroke Foundation
National Institute of General Medical SciencesR01GM015792
National Institute of General Medical Sciences


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