A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation


Huaying Zhao, National Institutes of Health
Rodolfo Ghirlando, National Institutes of Health
Carlos Alfonso, Centro de Investigaciones Biológicas
Fumio Arisaka, Nihon University
Ilan Attali, Tel Aviv University
David L. Bain, University of Colorado
Marina M. Bakhtina, Ohio State University
Donald F. Becker, University of Nebraska-Lincoln
Gregory J. Bedwell, University of Alabama
Ahmet Bekdemir, École Polytechnique Fédérale de Lausanne
Tabot M. D. Besong, Abdullah University of Science & Technology
Catherine Birck, Structural Biology Platform IGBMC
Chad A. Brautigam, University of Texas
William Brennerman, Beckman Coulter, Inc.
Olwyn Byron, University of Glasgow
Agnieszka Bzowska, University of Warsaw
Jonathan B. Chaires, University of Louisville
Catherine T. Chaton, University of Cincinnati
Helmut Colfen, University of Konstanz
Keith D. Connaghan, University of Colorado
Kimberly A. Crowley, University of Massachusetts
Ute Curth, Hannover Medical School
Tina Daviter, University of London
William L. Dean, University of Louisville
Ana I. Diez, University of Murcia
Christine Ebel, Univ. Grenoble Alpes
Debra M. Eckert, University of Utah
Leslie E. Eisele, Wadsworth Center
Edward Eisenstein, University of Maryland
Patrick England, Institut Pasteur
Carlos Escalante, Virginia Commonwealth University
Jeffrey A. Fagan, National Institute of Standards and Technology
Robert Fairman, Haverford College
Ron M. Finn, Max Planck Institute for Biophysical Chemistry
Wolfgang Fischle, Max Planck Institute for Biophysical Chemistry
Jose Garcia de la Torre, University of Murcia
Jayesh Gor, University College London
Henning Gustafsson, Novo Nordisk A/S
Damien Hall, Australian National University
Stephen E. Harding, University of Nottingham
Jose G. Hernandez Cifre, University of Murcia
Andrew B. Herr, University of Cincinnati
Elizabeth E. Howell, University of Tennessee
Richard S. Isaac, University of California
Shu-Chuan Jao, Academia Sinica
Davis Jose, University of Oregon
Soon-Jong Kim, Mokpo National University
Bashkim Kokona, Haverford College
Jack A. Komblatt, Concordia University
Dalibor Kosek, Charles University in Prague
Elena Krayukhina, Osaka University
Daniel Krzizike, Colorado State University
Eric A. Kusznir, Roche Innovation Center Basel
Hyewon Kwon, University of Washington
Adam Larson, University of California
Thomas M. Laue, University of New Hampshire
Aline Le Roy, Univ. Grenoble Alpes
Andrew P. Leech, University of York
Hauke Lilie, Martin-Luther University
Karolin Luger, Colorado State University
Juan R. Luque-Ortega, Centro de Investigaciones Biológicas
Jia Ma, National Institutes of Health
Carrie A. May, University of New Hampshire
Ernest L. Maynard, Uniformed Services University of the Health Sciences
Anna Modrak-Wojcik, University of Warsaw
Yee-Foong Mok, University of Melbourne
Norbert Mucke, German Cancer Research Center
Luitgard Nagel-Steger, Research Center Juelich
Geeta J. Narlikar, University of California
Masanori Noda, Osaka University
Amanda Nourse, St. Jude Children’s Research Hospital
Tomas Obsil, Charles University in Prague
Chad K. Park, University of Arizona
Jin-Ku Park, Mokpo National University
Peter D. Pawelek, Concordia University
Erby E. Perdue, Beckman Coulter, Inc.
Stephen J. Perkins, University College London
Matthew A. Perugini, La Trobe University
Craig L. Peterson, University of Massachusetts Medical School
Martin G. Peverelli, La Trobe University
Grzegorz Piszczek, National Institutes of Health
Gali Prag, Tel Aviv University
Peter E. Prevelige, University of Alabama
Bertrand D. E. Raynal, Institut Pasteur
Lenka Rezabkova, Paul Scherrer Institute
Klaus Richter, Technische Universität München
Alison E. Ringel, Johns Hopkins University School of Medicine
Rose Rosenberg, University of Konstanz
Arthur J. Rowe, University of Nottingham
Ame C. Rufer, Roche Innovation Center Basel
David J. Scott, Rutherford Appleton Laboratory
Javier G. Seravalli, University of Nebraska-Lincoln
Alexandra S. Solovyova, University of Newcastle
Renjie Song, New York State Department of Health
David Staunton, Oxford
Caitlin Stoddard, University of California
Katherine Stott, University of Cambridge
Holger M. Strauss, Novo Nordisk
Werner W. Streicher, Novo Nordisk
John P. Sumida, University of Washington
Sarah G. Swygert, University of Massachusetts Medical School
Roman H. Szczepanowski, International Institute of Molecular and Cell Biology
Ingrid Tessmer, University of Würzburg
Ronald T. Toth IV, University of Kansas
Ashutosh Tripathy, University of North Carolina
Susumu Uchiyama, Osaka University
Stephan F. W. Uebel, Max Planck Institute of Biochemistry
Satoru Unzai, Yokohama City University
Anna Vitlin Gruber, Tel Aviv University
Peter H. von Hippel, University of Oregon
Christine Wandrey, Ecole Polytechnique Fédérale de Lausanne
Szu-Huan Wang, Academia Sinica
Steven E. Weitzel, University of Oregon
Beata Wielgus-Kutrowska, University of Warsaw
Cynthia Wolberger, Johns Hopkins University School of Medicine
Martin Wolff, Research Center Juelich
Edward Wright, University of Tennessee
Yu-Sung Wu, University of Delaware
Jacinta M. Wubben, La Trobe University
Peter Schuck, National Institutes of Health


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.


Molecular, Cellular and Biomedical Sciences

Publication Date


Journal Title




Digital Object Identifier (DOI)

Document Type