34: Ultracentrifugation
- Page ID
- 142287
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Ultracentrifugation is used to separate and purify macromolecules, including nucleic acids, according to their respective densities. While the technique requires expensive equipment and is time-consuming, it is very sensitive to small changes in physical properties.
Also known as
High-speed centrifugation
Isopycnic centrifugation
Density gradient centrifugation
Samples needed
To conduct ultracentrifugation, you need:
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A mixture of DNA of different densities
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A centrifuge tube containing a cesium chloride salt gradient
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An ultracentrifuge
Method
To create the gradient, a mixture of DNA (or other macromolecule) of different densities is mixed with a cesium chloride (CsCl) solution and placed into a centrifuge tube. Upon centrifugation at extremely high speeds (>200,000 x g) in an ultracentrifuge, CsCl spontaneously dissociates into Cs+ and Cl- ions, and the heavier Cs+ ions move to the bottom of the tube. This movement is somewhat balanced by diffusion; therefore, the Cs+ ions form a continuous gradient with higher density at the bottom of the tube.
When a DNA mixture is placed in the tube and the tube is subjected to extremely high centrifugal forces, the DNA molecules will separate in the gradient according to their relative densities, with more dense DNA layering at the bottom of the tube. The tube can then be punctured and pure DNA of a specific density can be purified by extracting it with a needle/syringe. Ethidium bromide or another intercalating agent is often included during ultracentrifugation to enable visualization of the DNA bands under ultraviolet light.
Controls
While controls are not usually included in this separation/purification scheme, DNA markers of known density can be included for comparison purposes.
Interpretation
Figure 1. Virus purification by CsCl density gradient centrifugation. Relevant section of caption for published figure reads: “The crude suspensions of phages S13′ and φEF24C were purified by CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) and general centrifugation (40,000 × g, 2 h and 4 h, 4 °C). (a) Observation of virus bands. Black arrowheads indicate the phage bands. (b) Concentrations of phage bands. The phage band was collected from each tube, and phage concentration was measured. Bars indicate mean values with standard deviations. Statistical significance is indicated by asterisks (P < 0.05; n = 6).” “Figure 1" by Nasukawa et al.[1] [Image description]In part A of the figure, the bands corresponding to the S13′ and φEF24C phage strains following ultracentrifugation or general (lower-speed) centrifugation are shown in their centrifugation tubes (note: in this case, the bands likely contain both viral DNA and protein). The phage bands are indicated with black arrowheads and are distinctly separated from other nucleic acid bands in the mixture.
In part B of the figure, the number of phage particles was measured by calculating the number of plaque forming units (pfu) per milliliter of solution. Purification of the phage genomes by ultracentrifugation results in a concentration of phage about 1000-fold greater than that in a crude phage suspension, and equal to or greater than that obtained by general centrifugation at lower speeds. From these results, the authors concluded that regular centrifugation can be an effective method for purification of phage particles.
It should be noted that the duration of ultracentrifugation in these experiments (1 hour) was shorter than that of regular centrifugation. Typically, ultracentrifugation used to obtain extremely pure samples of specific nucleic acids is a long process, taking 12-24 hours.
Image Descriptions
Figure 1 image description:
Part A is a photograph of a set of centrifugation tubes containing phage DNA that has been subjected to centrifugation at both high and low speeds. The banding patterns in the tubes indicates that relatively pure populations of macromolecules have segregated by density to different positions in the tubes. Part B is bar graph showing the quantification of the number of phage in each of the bands. ↵
Thumbnail
"Beckman Model E Ultracentrifuge Rotor"↗ by Beckman Instruments, Inc is in the Public Domain↗.
Author
Mitch McVey, Tufts University
1. Nasukawa T., Uchiyama J., Taharaguchi S., Ota S., Ujihara T., Matsuzaki S., Murakami H., Mizukami K., and M Sakaguchi. Virus purification by CsCl density gradient using general centrifugation. 2017. Archives of Virology. 162: 3523–3528. ↵