Huaying Zhao, National Institute of Health
Rodolfo Ghirlando, National Institute of Health
Carlos Alfonso, Centro de Investigaciones Biologicas
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 - Birmingham
Ahmet Bekdemir, Ecole Polytechnique Federale de Lausanne
Tabot M. D. Besong, University of Nottingham, King Abdullah University
Catherine Birck, Structural Biology Platform IGBMC
Chad A. Brautigam, University of Texas Southwestern Medical Center at Dallas
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 Cölfen, University of Konstanz
Keith D. Connaghan, University of Colorado Anschutz Medical Campus
Kimberly A. Crowley, University of Massachusetts Medical School Worcester
Ute Curth, Hannover Medical School
Tina Daviter, University of London
William L. Dean, University of Louisville
Ana I. Díez, 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 UniversityFollow
Jeffrey A. Fagan, National Institute of Standards and Technology
Robert Fairman, Haverford College
Ron M. Finn, Laboratory of Chromatin Biochemistry
Wolfgang Fischle, Laboratory of Chromatin Biochemistry
José García 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
José G. Hernández Cifre, University of Murcia
Andrew B. Herr, University of Cincinnati
Elizabeth E. Howell, University of Tennessee - Knoxville
Richard S. Isaac, University of California - San Francisco
Shu-Chuan Jao, Academia Sinica
Davis Jose, University of Oregon
Soon-Jong Kim, Mokpo National University
Bashkim Kokona, Haverford College
Jack A. Kornblatt, Concordia University
Dalibor Kosek, Charles University, Prague
Elena Krayukhina, Osaka University
Daniel Krzizike, Colorado State University - Fort Collins
Eric A. Kusznir, Roche Innovation Center Basel, F. Hoffmann-LaRoche Ltd.
Hyewon Kwon, University of Washington - Seattle Campus
Adam Larson, University of California - San Francisco
Thomas M. Laue, University of New Hampshire
Aline Le Roy, Univ. Grenoble Alpes
Andrew P. Leech, University of York
Hauke Lilie, Matin-Luther University Halle-Wittenberg
Karolin Lugar, Colorado State University - Fort Collins
Juan R. Luque-Ortega, Centro de Investigaciones Biologicas
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 Mücke, German Cancer Research Center
Luitgard Nagel-Steger, Research Center Juelich
Geeta J. Narlikar, University of California - San Francisco
Masanori Noda, Osaka University
Amanda Nourse, St. Jude Children's Research Hospital
Tomas Obsil, Charles University, 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 Worcester
Martin G. Peverelli, La Trobe University
Grzegorz Piszczek, National Institutes of Health
Gali Prag, Tel Aviv University
Peter E. Prevelige, University of Alabama - Birmingham
Bertrand D. E. Raynal, Institut Pasteur
Lenka Rezabkova, Paul Scherrer Institute
Klaus Richter, Technische Universitat Munchen
Alison E. Ringel, Johns Hopkins University
Rose Rosenberg, University of Konstanz
Arthur J. Rowe, University of Nottingham
Arne C. Rufer, Roche Innovation Center Basel, F. Hoffmann-LaRoche Ltd.
David J. Scott, Research Complex at Harwell
Javier G. Seravalli, University of Nebraska-Lincoln
Alexandra S. Solovyova, University of Newcastle-upon-Tyne
Renjie Song, New York State Department of Health
David Staunton, Molecular Biophysics Suite
Catlin Stoddard, University of California - San Francisco
Katherine Stott, University of Cambridge
Holger M. Strauss, Novo Nordisk A/S
Werner W. Streicher, Novo Nordisk Foundation Center for Protein Research
John P. Sumida, University of Washington - Seattle Campus
Sarah G. Swygert, University of Massachusetts Medical School Worcester
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 at Chapel Hill
Susumu Uchiyama, Osaka University
Stephen F. W. Uebel, Max Planck Institut fur Biochemie
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
Martin Wolff, Research Center Juelich
Edward Wright, University of Tennessee - Knoxville
Yu-Sung Wu, University of Delaware
Jacinta M. Wubben, La Trobe University
Peter Schuck, National Institutes of HealthFollow

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November 2015


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.


This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication

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VCU Physiology and Biophysics Publications

S1_Fig.tif (202 kB)
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.