Analytical ultracentrifugation (AUC) is a first-principle centrifugation technique performed in a matrix-free environment. It eliminates the need for calibration standards and is unmatched by other methods in providing a comprehensive range of information about macromolecules with exceptional precision and accuracy.
One key application for AUC is determining molecular weight of proteins in solution, offering researchers valuable insights into molecule size, shape, and behavior. With sedimentation velocity, sedimentation-diffusion equilibrium and density gradient equilibrium measurements, AUC offers complementary perspectives on solution properties, including molar masses, stoichiometries, association constants, the cargo of drug carriers and more.
APPLICATIONS
Applications of AUC
Common AUC applications include:
- Extracellular vesicles
- Plasmids
- Nanoparticles
- Antibodies & conjugates
- Molecular Weight
- Protein Aggregation
- Stoichiometry
- Ligand Binding
- Viral Vectors
- Polydispersity
- Polymers
- Synthetics
FEATURES
Essential Features of the Optima Analytical Ultracentrifuge
A technique that has been involved in some of the most seminal discoveries in life sciences, AUC has made a major comeback with the introduction of the Optima AUC. This state-of-the-art instrument represents significant improvements in terms of usability, accuracy, precision, scan speed, temperature control, and much more.
The Optima AUC instrument uses two optical detection systems—absorbance measurements and Rayleigh-Interference—to monitor the behavior of macromolecules in real time and at various concentrations.
With its ability to characterize macromolecule/particle populations of heterogeneous samples, AUC is an indispensable tool for providing valuable insights into particle properties and their interactions. Specifically, among a wide variety of particles the Optima AUC ultracentrifuge can characterize are:
- Nanoparticles: AUC can be used to accurately determine size distribution of nanoparticles in a solution, providing orthogonal data (e.g., size, shape and density) to complement other analytical methods.
- Proteins: AUC analysis of proteins in their native state preserves their natural conformation and behavior, ensuring that data obtained accurately represents their true characteristics.
- Viruses/viral vectors: AUC is the only method with sufficient resolution and a large enough dynamic range to accurately quantify viral assembly states.
FAQs
How is an analytical ultracentrifuge different from a preparative ultracentrifuge?
An analytical ultracentrifuge differs from a preparative ultracentrifuge by incorporating an integrated optical detection system that allows real-time monitoring of particles in the sample as they undergo sedimentation. The optical systems, such as UV-Vis absorbance and Rayleigh interference, enable precise observation of the solution behavior of molecules. This real-time monitoring, coupled with advanced software, enables comprehensive analysis of molecules, providing valuable insights into their properties and behavior.
What types of molecules can be studied using AUC?
Most molecules can be studied using AUC, as long as they meet a few requirements. Specifically, they must be:
- Large enough to be moved in the centrifugal field; this would include macromolecules the size of peptides or larger
- Dissolved or dispersed in a liquid medium
- Unable to completely block light
Compared to other techniques, what are some advantages of using AUC for protein studies?
One key advantage is that AUC enables analysis of proteins in their native state, preserving their natural conformation and behavior in solution. Additionally, AUC provides a comprehensive analysis of proteins, including information on molecular weight, hydrodynamic properties, and solution behavior, all in a single experiment.
Another advantage comes from AUC being a first-principle technique: There is no need to use standards, and the results of AUC characterization are validated with data analysis.
The high resolution of AUC results from measuring two different independent thermodynamical parameters, the sedimentation coefficient and the diffusion coefficient of particle populations and using both of those parameters for biophysical characterization.
What type of information can be obtained from sedimentation velocity and sedimentation equilibrium experiments using AUC?
Sedimentation velocity experiments provide hydrodynamic information about the size and shape of molecules, as well as information about associations – all at concentrations close to the loading concentration.
In contrast, sedimentation-diffusion equilibrium experiments provide shape-independent insights into molar masses, stoichiometries, and association constants over a wide sample concentration range, and from one single experiment.
How does an analytical ultracentrifuge contribute to the study of biomolecular interactions?
The analytical ultracentrifuge plays a crucial role in studying biomolecular interactions by providing quantitative information about their association constants and stoichiometries. By analyzing the sedimentation behavior of macromolecules in solution, the analytical ultracentrifuge offers valuable insights into the strength and dynamics of biomolecular interactions, enabling researchers to understand molecular recognition processes and optimize the design of therapeutic agents or drug-target interactions.
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