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Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about...

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Autores principales: Balbo, Andrea, Zhao, Huaying, Brown, Patrick H., Schuck, Peter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157848/
https://www.ncbi.nlm.nih.gov/pubmed/19893484
http://dx.doi.org/10.3791/1530
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author Balbo, Andrea
Zhao, Huaying
Brown, Patrick H.
Schuck, Peter
author_facet Balbo, Andrea
Zhao, Huaying
Brown, Patrick H.
Schuck, Peter
author_sort Balbo, Andrea
collection PubMed
description The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions. This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows. This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.
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spelling pubmed-31578482011-08-19 Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell Balbo, Andrea Zhao, Huaying Brown, Patrick H. Schuck, Peter J Vis Exp Basic Protocols The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions. This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows. This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment. MyJove Corporation 2009-11-05 /pmc/articles/PMC3157848/ /pubmed/19893484 http://dx.doi.org/10.3791/1530 Text en Copyright © 2009, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Basic Protocols
Balbo, Andrea
Zhao, Huaying
Brown, Patrick H.
Schuck, Peter
Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell
title Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell
title_full Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell
title_fullStr Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell
title_full_unstemmed Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell
title_short Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell
title_sort assembly, loading, and alignment of an analytical ultracentrifuge sample cell
topic Basic Protocols
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157848/
https://www.ncbi.nlm.nih.gov/pubmed/19893484
http://dx.doi.org/10.3791/1530
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