Ultracentrifugation
Ultracentrifugation spins samples at a much higher speed than conventional centrifugation. Its rotation speed usually ranges from 60,000 to 150,000 rpm, and the centrifugal force from 200,000 to 1,000,000 g. This ultra-high speed allows for the differentiation of tiny density and size differences among pellets, viruses, organelles, proteins, and nucleic acids. Basically, the denser the component is, the faster it sediments in a certain centrifugal field.
Creative BioMart Nanocage ultracentrifugation services include both the preparative and analytical ultracentrifugation. The preparative ultracentrifugation service is desirable to attain small amounts of reference materials of recombinant protein nanocages with ultra purity as the standards in the following development process. Mechanisms of two types of preparative ultracentrifugations, gradient-density ultracentrifugation and differential ultracentrifugation are illustrated in the picture below. Analytical ultracentrifugation is designed to give reliable information about protein nanocage hydrodynamic and thermodynamic characteristics in solution.
Fig 1. Gradient-density Ultracentrifugation Mechanism (A) and Differential Ultracentrifugation Mechanism (B)(Jin, Y., et al., 2015, and Momen-Heravi, F., et al., 2013)
Preparative Ultracentrifugation Service
Preparative ultracentrifugation is mostly used to separate target components in a mixture. At Creative BioMart Nanocage, we offer density-gradient and differential ultracentrifugation. Density-gradient ultracentrifugation is equilibrium centrifugation. Samples are placed in a medium that forms a density gradient during the centrifugation. Components in the sample travel to and stay at the location where the density of the medium is the same as that of the component. Differential ultracentrifugation, by contrast, is rote-zonal centrifugation. It consists of a series of centrifugation with different centrifugal forces. After each centrifugation, sediments are separated and the remaining sample is subjected to a higher centrifugal force. Centrifugation over time will cause all components with higher density than the solvent to accumulate at the bottom of the centrifuge tube.
Table 1. Preparative ultracentrifugation service
| Type | Usages and features |
|---|---|
| Differential ultracentrifugation | Separation of organellse and other sub-cellular particles. |
| Sucrose density-gradient ultracentrifugation | Separation of particles with similar sizes but different densities, higher resolution than differential ultracentrifugation. Suitable for recombinant protein nanocage standard sample preparation. |
| CsCl density-gradient ultracentrifugation | Typically used for separation of DNA molecules and applicable in recombinant protein nanocage separation. Higher resolution than differential ultracentrifugation. |
Analytical Ultracentrifugation Service
Our analytical ultracentrifugation service is powerful and efficient in helping our customers to characterize the size, shape, mass, association state, and aggregation state of their protein nanocages. Analytical centrifuges are coupled with ultra-violet, refractive index or fluorescence detector for real-time molecular concentration monitoring. In analytical ultracentrifugation services, we offer sedimentation velocity (SV) and sedimentation equilibrium (SE) experiments. SV is a hydrodynamic method measuring the rate of molecules moving in a centrifugal field and recording the time course of the sedimentation process. SV is conducted at a very high speed. During the centrifugation, molecules form a boundary moving further away from the rotor center with time and eventually become pellets. The whole process is typically 4-6 h. SE is a thermodynamic method in which molecules are being studied in a steady equilibrium in solutions. This equilibrium is a balance between the centrifugal force that forces molecules to move outward and forms a concentration gradient across the centrifuge cell, and the diffusion of molecules that tries to eliminate the concentration gradient. The concentration distribution at equilibrium is measured and recorded. SE usually takes a couple of days to complete. It rotates samples at a relatively low speed in comparison with SV.
Table 2. Analytical ultracentrifugation service
| Type | Usages |
|---|---|
| Sedimentation velocity | Suitable for analysis of protein nanocage mass and size homogeneity, size distribution, aggregation amount, and overall shape, plus detection of protein conformational changes, as well as stoichiometry study of tight complexes between proteins. |
| Sedimentation equilibrium | Suitable for measuring molecular mass and studying protein-protein interactions, such as measuring the equilibrium constant of reversible association and the stoichiometry of complexes. |
Creative BioMart Nanocage is committed to a fast and reliable ultracentrifugation service. We own a variety of ultracentrifuges compatible with zonal rotors and swing bucket rotors holding centrifugal tubes of different volumes. We guarantee the high quality of protein nanocage standard samples from our preparative ultracentrifugation service and the characterization of hydrodynamic and thermodynamic properties of protein nanocages in analytical ultracentrifugation service. If you are interested in our ultracentrifugation service, please do not hesitate to contact us through online inquiry.
References
- Jin, Y., et al. (2015). "Ultracentrifugation-based multi-target affinity selection mass spectrometry." RSC advances, 5(130), 107616-107622.
- Momen-Heravi, F., et al. (2013). "Current methods for the isolation of extracellular vesicles." Biological chemistry, 394(10), 1253-1262.
- Lebowitz, J., et al. (2002). "Modern analytical ultracentrifugation in protein science: a tutorial review." Protein science,11(9), 2067-2079.
