Differential centrifugation

Differential centrifugation (also known as differential

velocity centrifugation)
centrifugation) is a very common procedure in
biochemistry and cell biology,
biology, which is used to separate organelles
and other sub-cellular particles based on their sedimentation rate.
rate.
Although often applied in biological analysis, differential
centrifugation is a general technique also suitable for crude
purification of non-living suspended particles (e.g. nanoparticles,
colloidal particles, viruses). In a typical case where differential
Differential centrifugation
centrifugation is used to analyze cell-biological phenomena (e.g.
organelle distribution), a tissue sample is first lysed to break the
cell membranes and release the organelles and cytosol. The lysate is then subjected to repeated
centrifugations, where particles that sediment sufficiently quickly at a given centrifugal force for a
given time form a compact "pellet" at the bottom of the centrifugation tube.[1]

After each centrifugation, the supernatant (non-pelleted solution) is removed from the tube and re-
centrifuged at an increased centrifugal force and/or time. Differential centrifugation is suitable for
crude separations on the basis of sedimentation rate, but more fine grained purifications may be done
on the basis of density through equilibrium density-gradient centrifugation.[2] Thus, the differential
centrifugation method is the successive pelleting of particles from the previous supernatant, using
increasingly higher centrifugation forces.[3] Cellular organelles separated by differential
centrifugation maintain a relatively high degree of normal functioning, as long as they are not subject
to denaturing conditions during isolation.[4][5]

Contents
Theory
Procedure
Ultracentrifugation
Differences between differential and density gradient centrifugation
See also
References

Theory
In a viscous fluid, the rate of sedimentation of a given suspended particle (as long as the particle is
denser than the fluid) is largely a function of the following factors:

Gravitational force
Difference in density
Fluid viscosity
 Particle size and shape

Larger particles sediment more quickly and at lower centrifugal forces. If a particle is less dense than
the fluid (e.g., fats in water), the particle will not sediment, but rather will float, regardless of strength
of the g-force experienced by the particle. Centrifugal force separates components not only on the
basis of density, but also of particle size and shape. In contrast, a more specialized equilibrium
density-gradient centrifugation produces a separation profile dependent on particle-density alone,
and therefore is suitable for more fine-grained separations.

High g-force makes sedimentation of small particles much faster than Brownian diffusion, even for
very small (nanoscale) particles. When a centrifuge is used, Stokes' law must be modified to account
for the variation in g-force with distance from the center of rotation.[6]

where

D is the minimum diameter of the particles expected to sediment (m)

η (or μ) is the fluid dynamic viscosity (Pa.s)
Rf is the final radius of rotation (m)
Ri is the initial radius of rotation (m)
ρp is particle volumetric mass density (kg/m3)
ρf is the fluid volumetric mass density (kg/m3)
ω is the angular velocity (radian/s)
t is the time required to sediment from Ri to Rf (s)

Procedure
Differential centrifugation can be used with intact particles (e.g. biological cells, microparticles,
nanoparticles), or used to separate the component parts of a given particle.[7] Using the example of a
separation of eukaryotic organelles from intact cells, the cell must first be lysed and homogenized
(ideally by a gentle technique, such as Dounce homogenization; harsher techniques or over
homogenization will lead to a lower proportion of intact organelles). Once the crude organelle extract
is obtained, it may be subjected to a varying centrifugation speeds to separate the organelles:
 Typical differential centrifugation parameters for a biological sample[2] (path length of centrifugation ≈1–5 cm)
Sample input G force Time Instrument needed Pellet contents Supernatant contents
Benchtop fixed-angle
Unlysed
5 centrifuge, or Intact (eukaryotic) cells, Varies depending on
(eukaryotic) 100 x g
min swinging bucket macroscopic debris sample
cells
centrifuge
Gently lysed Benchtop fixed-angle
cells (e.g. 10 centrifuge, or Cytosol, non-nuclei
600 x g Nuclei
dounce min swinging bucket organelles
homogenizer) centrifuge
Cytosol, microsomes
Supernatant of 15,000 20 Benchtop fixed-angle Mitochondria, chloroplasts, (known as post
previous row xg min centrifuge lysosomes, peroxisomes mitochondrial
supernatant)
50,000 High speed fixed- Cytosol, ribosomal
Plasma membrane,
Supernatant of xg- 60 angle centrifuge, or subunits, small
microsomal fraction, large
previous row 100,000 min vacuum polyribosomes, enzyme
polyribosomes
xg ultracentrifuge complexes
50,000
Ribosomal subunits, small
Supernatant of xg- 120 Vacuum
poly ribosomes, some Cytosol
previous row 100,000 min ultracentrifuge
soluble enzyme complexes
xg

Ultracentrifugation
The lysed sample is now ready for centrifugation in an ultracentrifuge. An ultracentrifuge consists of a
refrigerated, low-pressure chamber containing a rotor which is driven by an electrical motor capable
of high speed rotation. Samples are placed in tubes within or attached to the rotor. Rotational speed
may reach up to 100,000 rpm for floor model, 150,000 rpm for bench-top model (Beckman Optima
Max-XP or Sorvall MTX150 or himac CS150NX), creating centrifugal speed forces of 800,000g to
1,000,000g. This force causes sedimentation of macromolecules, and can even cause non-uniform
distributions of small molecules.[8]

Since different fragments of a cell have different sizes and densities, each fragment will settle into a
pellet with different minimum centrifugal forces. Thus, separation of the sample into different layers
can be done by first centrifuging the original lysate under weak forces, removing the pellet, then
exposing the subsequent supernatants to sequentially greater centrifugal fields. Each time a portion of
different density is sedimented to the bottom of the container and extracted, and repeated application
produces a rank of layers which includes different parts of the original sample. Additional steps can be
taken to further refine each of the obtained pellets.

Sedimentation depends on mass, shape, and partial specific volume of a macromolecule, as well as
solvent density, rotor size and rate of rotation. The sedimentation velocity can be monitored during
the experiment to calculate molecular weight.
Values of sedimentation coefficient (S) can be
calculated. Large values of S (faster sedimentation rate) correspond to larger molecular weight. Dense
particle sediments more rapidly. Elongated proteins have larger frictional coefficients, and sediment
more slowly to ensure accuracy.
[9]
Differences between differential and density gradient
centrifugation
The difference between differential and density gradient centrifugation techniques is that the latter
method uses solutions of different densities (e.g. sucrose, ficoll) or gels through which the sample
passes. This separates the sample into layers by relative density, based on the principle that molecules
settle down under a centrifugal force until they reach a medium with the density the same as
theirs.[10] The degree of separation or number of layers depends on the solution or gel. Differential
centrifugation, on the other hand, does not utilize a density gradient, and the centrifugation is taken
in increasing speeds. The different centrifugation speeds often create separation into not more than
two fractions, so the supernatant can be separated further in additional centrifugation steps. For that,
each step the centrifugation speed has to be increased until the desired particles are separated. In
contrast, the density gradient centrifugation is usually performed with just one centrifugation
speed.[11]

See also
Buoyant density ultracentrifugation
Jerome Vinograd

References
1. Ohlendieck, Kay; Harding, Stephen E. (19 April 2018). "Centrifugation and Ultracentrifugation".
Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology: 424–453.
doi:10.1017/9781316677056.014 (https://doi.org/10.1017%2F9781316677056.014).
ISBN 9781107162273.
2. Darnell, James; Baltimore, David; Matsudaira, Paul; Zipursky, S. Lawrence; Berk, Arnold; Lodish,
Harvey (2000). "Purification of Cells and Their Parts" (https://www.ncbi.nlm.nih.gov/books/NBK21
492/).
3. Griffith, Owen Mitch (2010). Practical Techniques for Centrifugal Separations – Application Guide
(https://thermofisher.co.nz/Uploads/file/Scientific/Applications/Equipment-Furniture/Practical-Tech
niques-for-Centrifugal-Separations.pdf) (PDF). Principles & Techniques of Biochemistry and
Molecular Biology. p. 1-27.
4. Gerald Karp (19 October 2009). Cell and Molecular Biology: Concepts and Experiments (https://b
ooks.google.com/books?id=arRGYE0GxRQC&q=%22Differential+centrifugation%22&pg=PR28).
John Wiley & Sons. pp. 28–. ISBN 978-0-470-48337-4.
5. Livshits, Mikhail A.; Khomyakova, Elena; Evtushenko, Evgeniy G.; Lazarev, Vassili N.; Kulemin,
Nikolay A.; Semina, Svetlana E.; Generozov, Edward V.; Govorun, Vadim M. (30 November 2015).
"Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used
protocol" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663484). Scientific Reports. 5 (1):
17319. Bibcode:2015NatSR...517319L (https://ui.adsabs.harvard.edu/abs/2015NatSR...517319L).
doi:10.1038/srep17319 (https://doi.org/10.1038%2Fsrep17319). ISSN 2045-2322 (https://www.wo
rldcat.org/issn/2045-2322). PMC 4663484 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC466348
4). PMID 26616523 (https://pubmed.ncbi.nlm.nih.gov/26616523). S2CID 14200669 (https://api.se
manticscholar.org/CorpusID:14200669).
6. Harding, Stephen E.; Scott, David; Rowe, Arther (16 December 2007). Analytical
Ultracentrifugation: Techniques and Methods (https://books.google.com/books?id=vm0oDwAAQB
AJ&pg=PA271). Royal Society of Chemistry. ISBN 978-1-84755-261-7.
 7. Frei, Mark. "Centrifugation Separations" (https://www.sigmaaldrich.com/technical-documents/articl
es/biofiles/centrifugation-separations.html). BioFiles. 6 (5): 6–7.
8. Taylor, Douglas D.; Shah, Sahil (1 October 2015). "Methods of isolating extracellular vesicles
impact down-stream analyses of their cargoes". Methods. 87: 3–10.
doi:10.1016/j.ymeth.2015.02.019 (https://doi.org/10.1016%2Fj.ymeth.2015.02.019).
PMID 25766927 (https://pubmed.ncbi.nlm.nih.gov/25766927).
9. Vance, Dennis E.; Vance, J. E. (6 August 1996). "Structure, assembly and secretion of
lipoproteins" (https://books.google.com/books?id=EGMuwXqDbkcC&q=%22Structure%2C+asse
mbly+and+secretion+of+lipoproteins%22&pg=PA473). Biochemistry of Lipids, Lipoproteins and
Membranes. Elsevier. ISBN 978-0-08-086092-3.
10. Sapkota, Anupama (3 September 2020). "Types of Centrifuge & Centrifugation (definition,
principle, uses)" (https://microbenotes.com/centrifuge-and-centrifugation/). Microbe Notes.
11. Yu, Li-Li; Zhu, Jing; Liu, Jin-Xia; Jiang, Feng; Ni, Wen-Kai; Qu, Li-Shuai; Ni, Run-Zhou; Lu, Cui-
Hua; Xiao, Ming-Bing (2018). "A Comparison of Traditional and Novel Methods for the Separation
of Exosomes from Human Samples" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6083592).
BioMed Research International. 2018: 1–9. doi:10.1155/2018/3634563 (https://doi.org/10.1155%2
F2018%2F3634563). PMC 6083592 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6083592).
PMID 30148165 (https://pubmed.ncbi.nlm.nih.gov/30148165).

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