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2. Evaluation of Tetrazine Tracers for Pretargeted Imaging within the Central Nervous System

Author

Edelmann, Martin R., primary, Bredack, Christoph, additional, Belli, Sara, additional, Mohr, Peter, additional, Imhoff, Marie-Paule, additional, Reggiani, Flore, additional, Kusznir, Eric A., additional, Rufer, Arne C., additional, Holt, Daniel P., additional, Valentine, Heather, additional, Wong, Dean F., additional, Dannals, Robert F., additional, Honer, Michael, additional, and Gobbi, Luca C., additional

Published
2023
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6. EFMC – Trends that Link Medicinal Chemistry and Chemical Biology to Translational Drug Discovery

Author

Auberson, Yves P., primary, Arimondo, Paola B., additional, Duca, Maria, additional, Essig, Sebastian, additional, Grether, Uwe, additional, Rufer, Arne C., additional, Sbardella, Gianluca, additional, Schopfer, Ulrich, additional, Torrens, Antoni, additional, van der Stelt, Mario, additional, Vauzeilles, Boris, additional, Vázquez, Olalla, additional, and Zhang, Andrew X., additional

Published
2023
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8. EFMC - trends that link medicinal chemistry and chemical biology to translational drug discovery

Author

Auberson, Yves P., Arimondo, Paola B., Duca, Maria, Essig, Sebastian, Grether, Uwe, Rufer, Arne C., Sbardella, Gianluca, Schopfer, Ulrich, Torrens, Antoni, van der Stelt, Mario, Vauzeilles, Boris, Vázquez, Olalla, and Zhang, Andrew X.

Abstract

Ground-breaking research in disease biology and continuous efforts in method development have uncovered a range of potential new drug targets. Increasingly, the drug discovery process is informed by technologies involving chemical probes as tools. Applications for chemical probes comprise target identification and assessment, as well as the qualification of small molecules as chemical starting points and drug candidates. Progress in probe chemistry has opened the way to novel assay formats and pharmaceutical compound classes. The European Federation of Medicinal Chemistry and Chemical Biology (EFMC) has launched the Chemical Biology Initiative to advance science in the field of medicinal chemistry and chemical biology, while representing all members of this extended scientific community. This review provides an overview of the many important developments in the field of chemical biology that have happened at the lively interface of academic and industrial research.

Published
2023

11. Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe

Author

Gazzi, Thais, primary, Brennecke, Benjamin, additional, Atz, Kenneth, additional, Korn, Claudia, additional, Sykes, David, additional, Forn-Cuni, Gabriel, additional, Pfaff, Patrick, additional, Sarott, Roman C., additional, Westphal, Matthias V., additional, Mostinski, Yelena, additional, Mach, Leonard, additional, Wasinska-Kalwa, Malgorzata, additional, Weise, Marie, additional, Hoare, Bradley L., additional, Miljuš, Tamara, additional, Mexi, Maira, additional, Roth, Nicolas, additional, Koers, Eline J., additional, Guba, Wolfgang, additional, Alker, André, additional, Rufer, Arne C., additional, Kusznir, Eric A., additional, Huber, Sylwia, additional, Raposo, Catarina, additional, Zirwes, Elisabeth A., additional, Osterwald, Anja, additional, Pavlovic, Anto, additional, Moes, Svenja, additional, Beck, Jennifer, additional, Nettekoven, Matthias, additional, Benito-Cuesta, Irene, additional, Grande, Teresa, additional, Drawnel, Faye, additional, Widmer, Gabriella, additional, Holzer, Daniela, additional, van der Wel, Tom, additional, Mandhair, Harpreet, additional, Honer, Michael, additional, Fingerle, Jürgen, additional, Scheffel, Jörg, additional, Broichhagen, Johannes, additional, Gawrisch, Klaus, additional, Romero, Julián, additional, Hillard, Cecilia J., additional, Varga, Zoltan V., additional, van der Stelt, Mario, additional, Pacher, Pal, additional, Gertsch, Jürg, additional, Ullmer, Christoph, additional, McCormick, Peter J., additional, Oddi, Sergio, additional, Spaink, Herman P., additional, Maccarrone, Mauro, additional, Veprintsev, Dmitry B., additional, Carreira, Erick M., additional, Grether, Uwe, additional, and Nazaré, Marc, additional

Published
2022
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18. Diverse chemotypes drive biased signaling by cannabinoid receptors

Author

Miljuš, Tamara, primary, Heydenreich, Franziska M., additional, Gazzi, Thais, additional, Kimbara, Atsushi, additional, Rogers-Evans, Mark, additional, Nettekoven, Matthias, additional, Zirwes, Elisabeth, additional, Osterwald, Anja, additional, Rufer, Arne C., additional, Ullmer, Christoph, additional, Guba, Wolfgang, additional, Le Gouill, Christian, additional, Fingerle, Jürgen, additional, Nazaré, Marc, additional, Grether, Uwe, additional, Bouvier, Michel, additional, and Veprintsev, Dmitry B., additional

Published
2020
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19. Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

Author

Sarott, Roman C., primary, Westphal, Matthias V., additional, Pfaff, Patrick, additional, Korn, Claudia, additional, Sykes, David A., additional, Gazzi, Thais, additional, Brennecke, Benjamin, additional, Atz, Kenneth, additional, Weise, Marie, additional, Mostinski, Yelena, additional, Hompluem, Pattarin, additional, Koers, Eline, additional, Miljuš, Tamara, additional, Roth, Nicolas J., additional, Asmelash, Hermon, additional, Vong, Man C., additional, Piovesan, Jacopo, additional, Guba, Wolfgang, additional, Rufer, Arne C., additional, Kusznir, Eric A., additional, Huber, Sylwia, additional, Raposo, Catarina, additional, Zirwes, Elisabeth A., additional, Osterwald, Anja, additional, Pavlovic, Anto, additional, Moes, Svenja, additional, Beck, Jennifer, additional, Benito-Cuesta, Irene, additional, Grande, Teresa, additional, Ruiz de Martı́n Esteban, Samuel, additional, Yeliseev, Alexei, additional, Drawnel, Faye, additional, Widmer, Gabriella, additional, Holzer, Daniela, additional, van der Wel, Tom, additional, Mandhair, Harpreet, additional, Yuan, Cheng-Yin, additional, Drobyski, William R., additional, Saroz, Yurii, additional, Grimsey, Natasha, additional, Honer, Michael, additional, Fingerle, Jürgen, additional, Gawrisch, Klaus, additional, Romero, Julian, additional, Hillard, Cecilia J., additional, Varga, Zoltan V., additional, van der Stelt, Mario, additional, Pacher, Pal, additional, Gertsch, Jürg, additional, McCormick, Peter J., additional, Ullmer, Christoph, additional, Oddi, Sergio, additional, Maccarrone, Mauro, additional, Veprintsev, Dmitry B., additional, Nazaré, Marc, additional, Grether, Uwe, additional, and Carreira, Erick M., additional

Published
2020
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20. Reply to Alarcon and Borroto: Small molecule AX-024 reduces T cell proliferation independently of CD3ε-Nck1 interaction at SH3.1

Author

Richter, Kirsten, primary, Rufer, Arne C., additional, Muller, Magali, additional, Burger, Dominique, additional, Casagrande, Fabio, additional, Grossenbacher, Tabea, additional, Huber, Sylwia, additional, Hug, Melanie N., additional, Koldewey, Philipp, additional, D'Osualdo, Andrea, additional, Schlatter, Daniel, additional, Stoll, Theodor, additional, and Rudolph, Markus G., additional

Published
2020
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21. Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

Author

Sarott, Roman, primary, Westphal, Matthias, primary, Pfaff, Patrick, primary, Korn, Claudia, primary, Sykes, David, primary, Thais, Gazzi,, primary, Brennecke, Benjamin, primary, Atz, Kenneth, primary, Weise, Marie, primary, Mostinski, Yelena, primary, Hompluem, Pattarin, primary, eline, koers, primary, Miljuš, Tamara, primary, Roth, Nicolas, primary, Asmelash, Hermon, primary, Vong, Man, primary, Piovesan, Jacopo, primary, Guba, Wolfgang, primary, Rufer, Arne C., primary, A. Kusznir, Eric, primary, Huber, Sylwia, primary, Raposo, Catarina, primary, Zirwes, Elisabeth A., primary, Osterwald, Anja, primary, Pavlovic, Anto, primary, Moes, Svenja, primary, Beck, Jennifer, primary, Benito-Cuesta, Irene, primary, Grande, Teresa, primary, ruiz de martin esteban, Samuel, primary, Yeliseev, Alexei, primary, Drawnel, Faye, primary, Widmer, Gabriella, primary, Holzer, Daniela, primary, Wel, Tom van der, primary, Mandhair, Harpreet, primary, Yuan, Chen-Yin, primary, Drobyski, Wiliam, primary, Saroz, Yurii, primary, Grimsey, Natasha, primary, Honer, Michael, primary, Fingerle, Jürgen, primary, Gawrisch, Klaus, primary, Romero, Julian, primary, Hillard, Cecilia J., primary, Varga, Zoltan V., primary, Stelt, Mario van der, primary, Pacher, Pal, primary, Gertsch, Jürg, primary, McCormick, Peter, primary, Ullmer, Christoph, primary, Oddi, Sergio, primary, Maccarrone, Mauro, primary, Veprintsev, Dmitry B., primary, Nazare, Marc, primary, Grether, Uwe, primary, and Carreira, Erick, primary

Published
2020
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22. Highly Specific, Fluorescent Cannabinoid Type 2 Receptor Probes Enable Applications in Microscopy, Flow Cytometry and FRET-based Binding Assays

Author

Sarott, Roman, primary, Westphal, Matthias, primary, Pfaff, Patrick, primary, Korn, Claudia, primary, Sykes, David, primary, Thais, Gazzi,, primary, Brennecke, Benjamin, primary, Atz, Kenneth, primary, Weise, Marie, primary, Mostinski, Yelena, primary, Hompluem, Pattarin, primary, Miljuš, Tamara, primary, Roth, Nicolas, primary, Asmelash, Hermon, primary, Vong, Man, primary, Guba, Wolfgang, primary, Rufer, Arne C., primary, A. Kusznir, Eric, primary, Huber, Sylwia, primary, Raposo, Catarina, primary, Zirwes, Elisabeth A., primary, Osterwald, Anja, primary, Pavlovic, Anto, primary, Moes, Svenja, primary, Beck, Jennifer, primary, Benito-Cuesta, Irene, primary, Grande, Teresa, primary, Yeliseev, Alexei, primary, Drawnel, Faye, primary, Widmer, Gabriella, primary, Holzer, Daniela, primary, Wel, Tom van der, primary, Mandhair, Harpreet, primary, Yuan, Chen-Yin, primary, Drobyski, Wiliam, primary, Saroz, Yurii, primary, Grimsey, Natasha, primary, Honer, Michael, primary, Fingerle, Jürgen, primary, Gawrisch, Klaus, primary, Romero, Julian, primary, Hillard, Cecilia J., primary, Varga, Zoltan V., primary, Stelt, Mario van der, primary, Pacher, Pal, primary, Gertsch, Jürg, primary, McCormick, Peter J., primary, Ullmer, Christoph, primary, Oddi, Sergio, primary, Maccarrone, Mauro, primary, Veprintsev, Dmitry B., primary, Nazare, Marc, primary, Grether, Uwe, primary, and Carreira, Erick, primary

Published
2019
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23. Drug Derived Fluorescent Probes for the Specific Visualization of Cannabinoid Type 2 Receptor - A Toolbox Approach

Author

Thais, Gazzi,, primary, Brennecke, Benjamin, primary, Atz, Kenneth, primary, Korn, Claudia, primary, Sykes, David, primary, Sarott, Roman C., primary, Westphal, Matthias V., primary, Pfaff, Patrick, primary, Weise, Marie, primary, Mostinski, Yelena, primary, Hoare, Bradley L., primary, Miljuš, Tamara, primary, Mexi, Maira, primary, Guba, Wolfgang, primary, Anker, André, primary, Rufer, Arne C., primary, A. Kusznir, Eric, primary, Huber, Sylwia, primary, Raposo, Catarina, primary, Zirwes, Elisabeth A., primary, Osterwald, Anja, primary, Pavlovic, Anto, primary, Moes, Svenja, primary, Beck, Jennifer, primary, Benito-Cuesta, Irene, primary, Grande, Teresa, primary, Drawnel, Faye, primary, Widmer, Gabriella, primary, Holzer, Daniela, primary, Wel, Tom van der, primary, Mandhair, Harpreet, primary, Saroz, Yurii, primary, Grimsey, Natasha, primary, Honer, Michael, primary, Fingerle, Jürgen, primary, Gawrisch, Klaus, primary, Romero, Julian, primary, Hillard, Cecilia J., primary, McCormick, Peter J., primary, Varga, Zoltan V., primary, Stelt, Mario van der, primary, Pacher, Pal, primary, Gertsch, Jürg, primary, Ullmer, Christoph, primary, Oddi, Sergio, primary, Maccarrone, Mauro, primary, Veprintsev, Dmitry B., primary, Carreira, Erick M., primary, Grether, Uwe, primary, and Nazare, Marc, primary

Published
2019
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27. Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody

Author

Favuzza, Paola, primary, Guffart, Elena, additional, Tamborrini, Marco, additional, Scherer, Bianca, additional, Dreyer, Anita M, additional, Rufer, Arne C, additional, Erny, Johannes, additional, Hoernschemeyer, Joerg, additional, Thoma, Ralf, additional, Schmid, Georg, additional, Gsell, Bernard, additional, Lamelas, Araceli, additional, Benz, Joerg, additional, Joseph, Catherine, additional, Matile, Hugues, additional, Pluschke, Gerd, additional, and Rudolph, Markus G, additional

Published
2017
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28. A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Author

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., Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., 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., Le Roy, Aline, 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, 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., and Schuck, Peter

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.

Published
2015

29. Author response: Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody

Author

Favuzza, Paola, primary, Guffart, Elena, additional, Tamborrini, Marco, additional, Scherer, Bianca, additional, Dreyer, Anita M, additional, Rufer, Arne C, additional, Erny, Johannes, additional, Hoernschemeyer, Joerg, additional, Thoma, Ralf, additional, Schmid, Georg, additional, Gsell, Bernard, additional, Lamelas, Araceli, additional, Benz, Joerg, additional, Joseph, Catherine, additional, Matile, Hugues, additional, Pluschke, Gerd, additional, and Rudolph, Markus G, additional

Published
2016
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30. Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Author

Plath, Friederike, primary, Ringler, Philippe, additional, Graff-Meyer, Alexandra, additional, Stahlberg, Henning, additional, Lauer, Matthias E., additional, Rufer, Arne C., additional, Graewert, Melissa A., additional, Svergun, Dmitri, additional, Gellermann, Gerald, additional, Finkler, Christof, additional, Stracke, Jan O., additional, Koulov, Atanas, additional, and Schnaible, Volker, additional

Published
2016
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31. Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones

Author

Banner, David W., primary, Gsell, Bernard, additional, Benz, Jörg, additional, Bertschinger, Julian, additional, Burger, Dominique, additional, Brack, Simon, additional, Cuppuleri, Simon, additional, Debulpaep, Maja, additional, Gast, Alain, additional, Grabulovski, Dragan, additional, Hennig, Michael, additional, Hilpert, Hans, additional, Huber, Walter, additional, Kuglstatter, Andreas, additional, Kusznir, Eric, additional, Laeremans, Toon, additional, Matile, Hugues, additional, Miscenic, Christian, additional, Rufer, Arne C., additional, Schlatter, Daniel, additional, Steyaert, Jan, additional, Stihle, Martine, additional, Thoma, Ralf, additional, Weber, Martin, additional, and Ruf, Armin, additional

Published
2013
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32. A Novel Selective Inverse Agonist of the CB2Receptor as a Radiolabeled Tool Compound for Kinetic Binding Studies

Author

Martella, Andrea, Sijben, Huub, Rufer, Arne C., Grether, Uwe, Fingerle, Juergen, Ullmer, Christoph, Hartung, Thomas, IJzerman, Adriaan P., van der Stelt, Mario, and Heitman, Laura H.

Abstract

The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [3H]CP55,940) are not optimal, despite the research efforts in developing effective drugs to target this system. At the same time, the importance of drug-target binding kinetics is growing since the kinetic binding profile of a ligand may provide important insights for the resulting in vivo efficacy. In this context we synthesized and characterized [3H]RO6957022, a highly selective CB2R inverse agonist, as a radiolabeled tool compound. In equilibrium and kinetic binding experiments [3H]RO6957022 showed high affinity for human CB2R with fast association (kon) and moderate dissociation (koff) kinetics. To demonstrate the robustness of [3H]RO6957022 binding, affinity studies were carried out for a wide range of CB2R reference ligands, spanning the range of full, partial, and inverse agonists. Finally, we used [3H]RO6957022 to study the kinetic binding profiles (i.e., konand koffvalues) of selected synthetic and endogenous (i.e., 2-arachidonoylglycerol, anandamide, and noladin ether) CB2R ligands by competition association experiments. All tested ligands, and in particular the endocannabinoids, displayed distinct kinetic profiles, shedding more light on their mechanism of action and the importance of association rates in the determination of CB2R affinity. Altogether, this study shows that the use of a novel tool compound, i.e., [3H]RO6957022, can support the development of novel ligands with a repertoire of kinetic binding profiles for CB2R.

Published
2017
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34. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Author

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 MD, 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, De La Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E, Cifre, José G Hernández, 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, Le Roy, Aline, 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 DE, 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 FW, 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, and Schuck, Peter

Subjects
Calibration, Reproducibility of Results, Ultracentrifugation, 3. Good health
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.

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

Author

Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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, Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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., and Schuck, Peter

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.

Full Text
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36. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Author

Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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, Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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., and Schuck, Peter

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.

Full Text
View/download PDF

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

Author

Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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, Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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., and Schuck, Peter

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.

Full Text
View/download PDF

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

Author

Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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, Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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., and Schuck, Peter

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.

Full Text
View/download PDF

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

Author

Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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, Langowski, Jörg, 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, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, 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., Le Roy, Aline, 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., and Schuck, Peter

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.

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40. Enhancing Drug Discovery and Development through the Integration of Medicinal Chemistry, Chemical Biology, and Academia-Industry Partnerships: Insights from Roche's Endocannabinoid System Projects.

Author

Aebi J, Atz K, Ametamey SM, Benz J, Blaising J, Butini S, Campiani G, Carreira EM, Collin L, De Lago E, Gazzi T, Gertsch J, Gobbi L, Guba W, Fernández-Ruiz J, Fingerle J, Haider A, He Y, Heitman LH, Honer M, Hunziker D, Kuhn B, Maccarrone M, Märki HP, Martin RE, Mohr P, Mu L, Nazaré M, Nippa DF, Oddi S, O'Hara F, Pacher P, Romero J, Röver S, Rufer AC, Schibli R, Schneider G, Stepan AF, Sykes DA, Ullmer C, Van der Stelt M, Veprintsev DB, Wittwer MB, and Grether U

Subjects
Humans, Drug Industry, Monoacylglycerol Lipases metabolism, Monoacylglycerol Lipases antagonists & inhibitors, Drug Development, Academia, Drug Discovery, Endocannabinoids metabolism, Endocannabinoids chemistry, Chemistry, Pharmaceutical
Abstract

The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples., (Copyright 2024 Johannes Aebi, Kenneth Atz, Simon M. Ametamey, Jörg Benz, Julie Blaising, Stefania Butini, Giuseppe Campiani, Erick M. Carreira, Ludovic Collin, Eva de Lago, Thais Gazzi, Jürg Gertsch, Luca Gobbi, Wolfgang Guba, Javier Fernández-Ruiz, Jürgen Fingerle, Ahmed Haider, Yingfang He, Laura H. Heitman, Michael Honer, Daniel Hunziker, Bernd Kuhn, Mauro Maccarrone, Hans Peter Märki, Rainer E. Martin, Peter Mohr, Linjing Mu, Marc Nazaré, David F. Nippa, Sergio Oddi, Fionn O’Hara, Pal Pacher, Julian Romero, Stephan Röver, Arne C. Rufer, Roger Schibli, Gisbert Schneider, Antonia F. Stepan, David A. Sykes, Christoph Ullmer, Mario van der Stelt, Dmitry B. Veprintsev, Matthias B. Wittwer, Uwe Grether. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

Published
2024
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41. A Novel Selective Inverse Agonist of the CB 2 Receptor as a Radiolabeled Tool Compound for Kinetic Binding Studies.

Author

Martella A, Sijben H, Rufer AC, Grether U, Fingerle J, Ullmer C, Hartung T, IJzerman AP, van der Stelt M, and Heitman LH

Subjects
Animals, CHO Cells, Cannabinoids pharmacology, Cricetinae, Cricetulus, Cyclohexanols metabolism, Cyclohexanols pharmacology, Dose-Response Relationship, Drug, Humans, Protein Binding physiology, Tritium metabolism, Cannabinoids agonists, Cannabinoids metabolism, Drug Inverse Agonism, Receptor, Cannabinoid, CB2 agonists, Receptor, Cannabinoid, CB2 metabolism
Abstract

The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [ 3 H]CP55,940) are not optimal, despite the research efforts in developing effective drugs to target this system. At the same time, the importance of drug-target binding kinetics is growing since the kinetic binding profile of a ligand may provide important insights for the resulting in vivo efficacy. In this context we synthesized and characterized [ 3 H]RO6957022, a highly selective CB2R inverse agonist, as a radiolabeled tool compound. In equilibrium and kinetic binding experiments [ 3 H]RO6957022 showed high affinity for human CB2R with fast association ( k on ) and moderate dissociation ( k off ) kinetics. To demonstrate the robustness of [ 3 H]RO6957022 binding, affinity studies were carried out for a wide range of CB2R reference ligands, spanning the range of full, partial, and inverse agonists. Finally, we used [ 3 H]RO6957022 to study the kinetic binding profiles (i.e., k on and k off values) of selected synthetic and endogenous (i.e., 2-arachidonoylglycerol, anandamide, and noladin ether) CB2R ligands by competition association experiments. All tested ligands, and in particular the endocannabinoids, displayed distinct kinetic profiles, shedding more light on their mechanism of action and the importance of association rates in the determination of CB2R affinity. Altogether, this study shows that the use of a novel tool compound, i.e., [ 3 H]RO6957022, can support the development of novel ligands with a repertoire of kinetic binding profiles for CB2R., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Published
2017
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42. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Author

Zhao H, Ghirlando R, Alfonso C, Arisaka F, Attali I, Bain DL, Bakhtina MM, Becker DF, Bedwell GJ, Bekdemir A, Besong TM, 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, de la Torre JG, Gor J, Gustafsson H, Hall D, Harding SE, 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, Le Roy A, 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 BD, 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 4th, Tripathy A, Uchiyama S, Uebel SF, 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, and Schuck P

Subjects
Calibration, Reproducibility of Results, Ultracentrifugation methods, Ultracentrifugation standards
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.

Published
2015
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