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. Connaghan & 100 others Kimberly 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

Research output: Contribution to journalArticle

  • 27 Citations

Abstract

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.

LanguageEnglish (US)
Article numbere0126420
JournalPloS one
Volume10
Issue number5
DOIs
StatePublished - May 21 2015

Fingerprint

ultracentrifugation
Ultracentrifugation
Calibration
bovine serum albumin
Bovine Serum Albumin
calibration
Molar mass
Sedimentation
Temperature
Monomers
molecular weight
Optical Devices
temperature
analytical kits
Centrifugation
Macromolecules
Optical systems
Dimers
centrifugation
methodology

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Zhao, H., Ghirlando, R., Alfonso, C., Arisaka, F., Attali, I., Bain, D. L., ... Schuck, P. (2015). A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. PloS one, 10(5), [e0126420]. DOI: 10.1371/journal.pone.0126420

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

In: PloS one, Vol. 10, No. 5, e0126420, 21.05.2015.

Research output: Contribution to journalArticle

Zhao, H, Ghirlando, R, Alfonso, C, Arisaka, F, Attali, I, Bain, DL, Bakhtina, MM, Becker, DF, Bedwell, GJ, Bekdemir, A, Besong, TMD, Birck, C, Brautigam, CA, Brennerman, W, Byron, O, Bzowska, A, Chaires, JB, Chaton, CT, Cölfen, H, Connaghan, KD, Crowley, KA, Curth, U, Daviter, T, Dean, WL, Díez, AI, Ebel, C, Eckert, DM, Eisele, LE, Eisenstein, E, England, P, Escalante, C, Fagan, JA, Fairman, R, Finn, RM, Fischle, W, García De La Torre, J, Gor, J, Gustafsson, H, Hall, D, Harding, SE, Hernández Cifre, JG, Herr, AB, Howell, EE, Isaac, RS, Jao, SC, Jose, D, Kim, SJ, Kokona, B, Kornblatt, JA, Kosek, D, Krayukhina, E, Krzizike, D, Kusznir, EA, Kwon, H, Larson, A, Laue, TM, Roy, AL, Leech, AP, Lilie, H, Luger, K, Luque-Ortega, JR, Ma, J, May, CA, Maynard, EL, Modrak-Wojcik, A, Mok, YF, Mücke, N, Nagel-Steger, L, Narlikar, GJ, Noda, M, Nourse, A, Obsil, T, Park, CK, Park, JK, Pawelek, PD, Perdue, EE, Perkins, SJ, Perugini, MA, Peterson, CL, Peverelli, MG, Piszczek, G, Prag, G, Prevelige, PE, Raynal, BDE, Rezabkova, L, Richter, K, Ringel, AE, Rosenberg, R, Rowe, AJ, Rufer, AC, Scott, DJ, Seravalli, JG, Solovyova, AS, Song, R, Staunton, D, Stoddard, C, Stott, K, Strauss, HM, Streicher, WW, Sumida, JP, Swygert, SG, Szczepanowski, RH, Tessmer, I, Toth, RT, Tripathy, A, Uchiyama, S, Uebel, SFW, Unzai, S, Gruber, AV, Von Hippel, PH, Wandrey, C, Wang, SH, Weitzel, SE, Wielgus-Kutrowska, B, Wolberger, C, Wolff, M, Wright, E, Wu, YS, Wubben, JM & Schuck, P 2015, 'A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation' PloS one, vol. 10, no. 5, e0126420. DOI: 10.1371/journal.pone.0126420
Zhao H, Ghirlando R, Alfonso C, Arisaka F, Attali I, Bain DL et al. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. PloS one. 2015 May 21;10(5). e0126420. Available from, DOI: 10.1371/journal.pone.0126420
Zhao, Huaying ; Ghirlando, Rodolfo ; Alfonso, Carlos ; Arisaka, Fumio ; Attali, Ilan ; Bain, David L. ; Bakhtina, Marina M. ; Becker, Donald F. ; Bedwell, Gregory J. ; Bekdemir, Ahmet ; Besong, Tabot M.D. ; Birck, Catherine ; Brautigam, Chad A. ; Brennerman, William ; Byron, Olwyn ; Bzowska, Agnieszka ; Chaires, Jonathan B. ; Chaton, Catherine T. ; Cölfen, Helmut ; Connaghan, Keith D. ; Crowley, Kimberly A. ; Curth, Ute ; Daviter, Tina ; Dean, William L. ; Díez, Ana I. ; Ebel, Christine ; Eckert, Debra M. ; Eisele, Leslie E. ; Eisenstein, Edward ; England, Patrick ; Escalante, Carlos ; Fagan, Jeffrey A. ; Fairman, Robert ; Finn, Ron M. ; Fischle, Wolfgang ; García De La Torre, José ; Gor, Jayesh ; Gustafsson, Henning ; Hall, Damien ; Harding, Stephen E. ; Hernández Cifre, José G. ; Herr, Andrew B. ; Howell, Elizabeth E. ; Isaac, Richard S. ; Jao, Shu Chuan ; Jose, Davis ; Kim, Soon Jong ; Kokona, Bashkim ; Kornblatt, Jack A. ; Kosek, Dalibor ; Krayukhina, Elena ; Krzizike, Daniel ; Kusznir, Eric A. ; Kwon, Hyewon ; Larson, Adam ; Laue, Thomas M. ; Roy, Aline Le ; Leech, Andrew P. ; Lilie, Hauke ; Luger, Karolin ; Luque-Ortega, Juan R. ; Ma, Jia ; May, Carrie A. ; Maynard, Ernest L. ; Modrak-Wojcik, Anna ; Mok, Yee Foong ; Mücke, Norbert ; Nagel-Steger, Luitgard ; Narlikar, Geeta J. ; Noda, Masanori ; Nourse, Amanda ; Obsil, Tomas ; Park, Chad K. ; Park, Jin Ku ; Pawelek, Peter D. ; Perdue, Erby E. ; Perkins, Stephen J. ; Perugini, Matthew A. ; Peterson, Craig L. ; Peverelli, Martin G. ; Piszczek, Grzegorz ; Prag, Gali ; Prevelige, Peter E. ; Raynal, Bertrand D.E. ; Rezabkova, Lenka ; Richter, Klaus ; Ringel, Alison E. ; Rosenberg, Rose ; Rowe, Arthur J. ; Rufer, Arne C. ; Scott, David J. ; Seravalli, Javier G. ; Solovyova, Alexandra S. ; Song, Renjie ; Staunton, David ; Stoddard, Caitlin ; Stott, Katherine ; Strauss, Holger M. ; Streicher, Werner W. ; Sumida, John P. ; Swygert, Sarah G. ; Szczepanowski, Roman H. ; Tessmer, Ingrid ; Toth, Ronald T. ; Tripathy, Ashutosh ; Uchiyama, Susumu ; Uebel, Stephan F.W. ; Unzai, Satoru ; Gruber, Anna Vitlin ; Von Hippel, Peter H. ; Wandrey, Christine ; Wang, Szu Huan ; Weitzel, Steven E. ; Wielgus-Kutrowska, Beata ; Wolberger, Cynthia ; Wolff, Martin ; Wright, Edward ; Wu, Yu Sung ; Wubben, Jacinta M. ; Schuck, Peter. / A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. In: PloS one. 2015 ; Vol. 10, No. 5.
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title = "A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation",
abstract = "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.",
author = "Huaying Zhao and Rodolfo Ghirlando and Carlos Alfonso and Fumio Arisaka and Ilan Attali and Bain, {David L.} and Bakhtina, {Marina M.} and Becker, {Donald F.} and Bedwell, {Gregory J.} and Ahmet Bekdemir and Besong, {Tabot M.D.} and Catherine Birck and Brautigam, {Chad A.} and William Brennerman and Olwyn Byron and Agnieszka Bzowska and Chaires, {Jonathan B.} and Chaton, {Catherine T.} and Helmut C{\"o}lfen and Connaghan, {Keith D.} and Crowley, {Kimberly A.} and Ute Curth and Tina Daviter and Dean, {William L.} and D{\'i}ez, {Ana I.} and Christine Ebel and Eckert, {Debra M.} and Eisele, {Leslie E.} and Edward Eisenstein and Patrick England and Carlos Escalante and Fagan, {Jeffrey A.} and Robert Fairman and Finn, {Ron M.} and Wolfgang Fischle and {Garc{\'i}a De La Torre}, Jos{\'e} and Jayesh Gor and Henning Gustafsson and Damien Hall and Harding, {Stephen E.} and {Hern{\'a}ndez Cifre}, {Jos{\'e} G.} and Herr, {Andrew B.} and Howell, {Elizabeth E.} and Isaac, {Richard S.} and Jao, {Shu Chuan} and Davis Jose and Kim, {Soon Jong} and Bashkim Kokona and Kornblatt, {Jack A.} and Dalibor Kosek and Elena Krayukhina and Daniel Krzizike and Kusznir, {Eric A.} and Hyewon Kwon and Adam Larson and Laue, {Thomas M.} and Roy, {Aline Le} and Leech, {Andrew P.} and Hauke Lilie and Karolin Luger and Luque-Ortega, {Juan R.} and Jia Ma and May, {Carrie A.} and Maynard, {Ernest L.} and Anna Modrak-Wojcik and Mok, {Yee Foong} and Norbert M{\"u}cke and Luitgard Nagel-Steger and Narlikar, {Geeta J.} and Masanori Noda and Amanda Nourse and Tomas Obsil and Park, {Chad K.} and Park, {Jin Ku} and Pawelek, {Peter D.} and Perdue, {Erby E.} and Perkins, {Stephen J.} and Perugini, {Matthew A.} and Peterson, {Craig L.} and Peverelli, {Martin G.} and Grzegorz Piszczek and Gali Prag and Prevelige, {Peter E.} and Raynal, {Bertrand D.E.} and Lenka Rezabkova and Klaus Richter and Ringel, {Alison E.} and Rose Rosenberg and Rowe, {Arthur J.} and Rufer, {Arne C.} and Scott, {David J.} and Seravalli, {Javier G.} and Solovyova, {Alexandra S.} and Renjie Song and David Staunton and Caitlin Stoddard and Katherine Stott and Strauss, {Holger M.} and Streicher, {Werner W.} and Sumida, {John P.} and Swygert, {Sarah G.} and Szczepanowski, {Roman H.} and Ingrid Tessmer and Toth, {Ronald T.} and Ashutosh Tripathy and Susumu Uchiyama and Uebel, {Stephan F.W.} and Satoru Unzai and Gruber, {Anna Vitlin} and {Von Hippel}, {Peter H.} and Christine Wandrey and Wang, {Szu Huan} and Weitzel, {Steven E.} and Beata Wielgus-Kutrowska and Cynthia Wolberger and Martin Wolff and Edward Wright and Wu, {Yu Sung} and Wubben, {Jacinta M.} and Peter Schuck",
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T1 - A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

AU - Zhao,Huaying

AU - Ghirlando,Rodolfo

AU - Alfonso,Carlos

AU - Arisaka,Fumio

AU - Attali,Ilan

AU - Bain,David L.

AU - Bakhtina,Marina M.

AU - Becker,Donald F.

AU - Bedwell,Gregory J.

AU - Bekdemir,Ahmet

AU - Besong,Tabot M.D.

AU - Birck,Catherine

AU - Brautigam,Chad A.

AU - Brennerman,William

AU - Byron,Olwyn

AU - Bzowska,Agnieszka

AU - Chaires,Jonathan B.

AU - Chaton,Catherine T.

AU - Cölfen,Helmut

AU - Connaghan,Keith D.

AU - Crowley,Kimberly A.

AU - Curth,Ute

AU - Daviter,Tina

AU - Dean,William L.

AU - Díez,Ana I.

AU - Ebel,Christine

AU - Eckert,Debra M.

AU - Eisele,Leslie E.

AU - Eisenstein,Edward

AU - England,Patrick

AU - Escalante,Carlos

AU - Fagan,Jeffrey A.

AU - Fairman,Robert

AU - Finn,Ron M.

AU - Fischle,Wolfgang

AU - García De La Torre,José

AU - Gor,Jayesh

AU - Gustafsson,Henning

AU - Hall,Damien

AU - Harding,Stephen E.

AU - Hernández Cifre,José G.

AU - Herr,Andrew B.

AU - Howell,Elizabeth E.

AU - Isaac,Richard S.

AU - Jao,Shu Chuan

AU - Jose,Davis

AU - Kim,Soon Jong

AU - Kokona,Bashkim

AU - Kornblatt,Jack A.

AU - Kosek,Dalibor

AU - Krayukhina,Elena

AU - Krzizike,Daniel

AU - Kusznir,Eric A.

AU - Kwon,Hyewon

AU - Larson,Adam

AU - Laue,Thomas M.

AU - Roy,Aline Le

AU - Leech,Andrew P.

AU - Lilie,Hauke

AU - Luger,Karolin

AU - Luque-Ortega,Juan R.

AU - Ma,Jia

AU - May,Carrie A.

AU - Maynard,Ernest L.

AU - Modrak-Wojcik,Anna

AU - Mok,Yee Foong

AU - Mücke,Norbert

AU - Nagel-Steger,Luitgard

AU - Narlikar,Geeta J.

AU - Noda,Masanori

AU - Nourse,Amanda

AU - Obsil,Tomas

AU - Park,Chad K.

AU - Park,Jin Ku

AU - Pawelek,Peter D.

AU - Perdue,Erby E.

AU - Perkins,Stephen J.

AU - Perugini,Matthew A.

AU - Peterson,Craig L.

AU - Peverelli,Martin G.

AU - Piszczek,Grzegorz

AU - Prag,Gali

AU - Prevelige,Peter E.

AU - Raynal,Bertrand D.E.

AU - Rezabkova,Lenka

AU - Richter,Klaus

AU - Ringel,Alison E.

AU - Rosenberg,Rose

AU - Rowe,Arthur J.

AU - Rufer,Arne C.

AU - Scott,David J.

AU - Seravalli,Javier G.

AU - Solovyova,Alexandra S.

AU - Song,Renjie

AU - Staunton,David

AU - Stoddard,Caitlin

AU - Stott,Katherine

AU - Strauss,Holger M.

AU - Streicher,Werner W.

AU - Sumida,John P.

AU - Swygert,Sarah G.

AU - Szczepanowski,Roman H.

AU - Tessmer,Ingrid

AU - Toth,Ronald T.

AU - Tripathy,Ashutosh

AU - Uchiyama,Susumu

AU - Uebel,Stephan F.W.

AU - Unzai,Satoru

AU - Gruber,Anna Vitlin

AU - Von Hippel,Peter H.

AU - Wandrey,Christine

AU - Wang,Szu Huan

AU - Weitzel,Steven E.

AU - Wielgus-Kutrowska,Beata

AU - Wolberger,Cynthia

AU - Wolff,Martin

AU - Wright,Edward

AU - Wu,Yu Sung

AU - Wubben,Jacinta M.

AU - Schuck,Peter

PY - 2015/5/21

Y1 - 2015/5/21

N2 - 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.

AB - 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.

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