Analytical ultracentrifugation
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Analytical ultracentrifugation is an analytical technique which combines an
ultracentrifuge with optical monitoring systems.
Analytical Ultracentrifuge. This appears to be a Spinco Model E. This is an early instrument, probably
dating to the 1950's. The operator is seated in front of the sample chamber, with his left hand touching the
rotor. In operation, the chamber would be sealed behind an armored shroud and pumped down to vacuum.
The shroud has been lowered to allow access for loading or unloading the rotor.[1]
In an analytical ultracentrifuge (commonly abbreviated as AUC), a sample’s
sedimentation profile is monitored in real time by an optical detection system. The
sample is detected via ultraviolet light absorption and/or interference optical
refractive index sensitive system. The operator can thus observe the evolution of
sample concentration versus the axis of the rotation profile as a result of the applied
centrifugal field. With modern instrumentation, these observations are electronically
digitized and stored for further mathematical analysis.
The information that can be obtained from an analytical ultracentrifuge includes the
gross shape of macromolecules, conformational changes in macromolecules, and
size distributions of macromolecules. With AUC it is possible to gain information on
the number and subunit stoichiometry of non-covalent complexes and equilibrium
constant constants of macromolecules such as proteins, DNA, nanoparticles or other
assemblies from different molecule classes.
Analytical ultracentrifugation has recently seen a rise in use because of increased
ease of analysis with modern computers and the development of software, including
a National Institutes of Health supported software package, SedFit.
Contents
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1History
2Instrumentation
3Theory
4Types of experiments
o 4.1Sedimentation velocity
o 4.2Sedimentation equilibrium
o 4.3Density gradient centrifugation
5Data evaluation
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6See also
7References
8External links
9Further reading
History[edit]
Instrumentation[edit]
Rotors for an Analytical Ultracentrifuge for a maximal spinning velocity of 50'000 (left) and 60'000 (right)
rpm
An analytical ultracentrifuge extends the ultracentrifuge by a light source and optical
detectors. To allow the light pass through the analyte during the ultracentrifuge run,
specialized cells are required which have to meet optical qualities as well as to resist
the gravitational forces. Each cell consists of a housing, two windows from quartz
glass, a center-piece with two sectors. These cell are placed into a rotor with
continuous bore.
Theory[edit]
Types of experiments[edit]
By applying specific equipment and adapting measurement parameters several
types of experiments can be performed. Most common AUC experiments are
sedimentation velocity and sedimentation equilibrium experiments.
Sedimentation velocity[edit]
Sedimentation velocity experiments render the shape and molar mass of the
analytes, as well as their size-distribution.[2] The size resolution of this method scales
approximately with the square of the particle radii, and by adjusting the rotor speed
of the experiment size-ranges from 100 Da to 10 GDa can be covered.
Sedimentation velocity experiments can also be used to study reversible chemical
equilibria between macromolecular species, by either monitoring the number and
molar mass of macromolecular complexes, by gaining information about the complex
composition from multi-signal analysis exploiting differences in each components
spectroscopic signal, or by following the composition dependence of the
sedimentation rates of the macromolecular system, as described in Gilbert-Jenkins
theory.
The experiment aims to monitor the sedimentation behavior at a fixed angular speed.
Sedimentation equilibrium[edit]
Sedimentation equilibrium experiments reports on the molar mass of analytes and
on chemical equilibrium constants. [3]
They are concerned with adjusting the rotor speed such that a steady-state
concentration profile c(r) of the sample in the cell is formed, where sedimentation
and diffusion cancel out each other.
Density gradient centrifugation[edit]
Data evaluation[edit]
See also[edit]
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Ultracentrifuge
Gas centrifuge
Theodor Svedberg
Differential centrifugation
Buoyant density ultracentrifugation
Zippe-type centrifuge
References[edit]
1.
2.
3.
^ "Technical Manual, Spinco Ultracentrifuge Model E". Science History Institute Digital
Collections. Retrieved 2018-12-18.
^ Perez-Ramirez, B. and Steckert, J.J. (2005). Therapeutic Proteins: Methods and Protocols. C.M.
Smales and D.C. James, Eds. Volume 308: 301-318. Humana Press Inc, Totowa, NJ.
^ Ghirlando, R. (2011). "The analysis of macromolecular interactions by sedimentation
equilibrium". Modern Analytical Ultracentrifugation: Methods. 58 (1): 145–
156. doi:10.1016/j.ymeth.2010.12.005. PMC 3090454. PMID 21167941.
External links[edit]
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Reversible Associations in Structural and Molecular Biology (RASMB -an
Analytical Ultracentrifugation Forum)
Analytical Ultracentrifugation as a Contemporary Biomolecular Research Tool.
Gilbert-Jenkins theory
Report on an ultracentrifuge explosion.
Further reading[edit]
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Lebowitz J, Lewis MS, Schuck P (Jan 2005). "Modern analytical ultracentrifugation in protein science:
A tutorial review". Protein Science. 102: 2067–
2069. doi:10.1110/ps.0207702. PMC 2373601. PMID 12192063.
Balbo A, Minor KH, Velikovsky CA, Mariuzza RA, Peterson CB, Schuck P (Jan 2005). "Studying
multiprotein complexes by multisignal sedimentation velocity analytical
ultracentrifugation". PNAS. 102 (1): 81–86. doi:10.1073/pnas.0408399102.
Cole J, Hansen J (Dec 1999). "Analytical ultracentrifugation as a contemporary biomolecular research
tool". J Biomol Tech. 10 (4). PMID 19499023.
Categories:
Centrifuges
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Analytical chemistry
Laboratory techniques
Biophysics
Fluid dynamics