Centrifugation

=Centrifugation =

Centrifugation is a technique in which components are separated by size using centripetal force.
 * Basic Description: **[[image:http://3.bp.blogspot.com/_xW3FQUQ2DYI/Rp4DF1r_0HI/AAAAAAAAAhY/B5MzdxVSV6I/s400/centrifugation.png align="right"]]

**Purpose of this technique:** The purpose of this technique is to separate cell components by size and density. The high speeds cause centripetal force and forces the components to separate and form a pellet at the bottom of the centrifuge tube. In general, the largest molecules feel the most centripetal force and move more rapidly than the other smaller, less dense molecules. Therefore, the components that form a pellet at the bottom of the centrifuge tube depending on their speeds in order from slowest to fastest speeds are nuclei sediment, mitochondria, small closed vesicles, and ribosomes (Alberts, 2002). Generally, the smaller the cellular component that needs isolation, the faster the centrifuge must rotate. In order to get the smallest components, the centrifugation must occur over several steps, by removing the pellet and re-centrifuging the supernatant (Alberts 2008).

Nuclei Cytoskeletons || Lysosomes Peroxisomes || Small Vesicles || Viruses Large Macromolecules || *The organelles obtained column refers to organelles in pellet **In order to collect the smallest organelles, slower centrifugations must be done to remove larger molecules first.**
 * **Force (g)** || **Time (min)** || **Organelles Obtained** ||
 * 1000 || 10 || Whole Cells
 * 20000 || 20 || Mitochondria
 * 80000 || 60 || Microsomes
 * 150000 || 180 || Ribosomes


 * Present-day ultracentrifuges rotate at speeds up to 80,000 rpm and produce forces 500,000 times greater than gravity. The ultracentrifuge is also used to separate cellular components based on their buoyant density, independent of their size and shape. In this case the sample is usually sedimented through a steep gradient and as each component reaches a position where the density of the solution and the component are equal the component stops and floats in that position. This creates a series of bands with the bands closest to the bottom of the tube containing the highest buoyancy density. This method is called equilibrium sedimentation. This method can also separate macromolecules that contain heavy isotopes from the same macromolecules with lighter isotopes (Alberts, 2002). **

Origin and History:


 * The history of the centrifuge was developed first not for scientific purposes but to separate cream from milk. Early devices were hand driven machines that spun. The first centrifuge can be traced back to the 1400’s. In 1864 Antonin Prandtl commercialized the centrifuge again for separating cream from milk. **
 * In 1926 Svedberg developed the first analytical ultracentrifuge and used it to estimate mass of hemoglobin as 68,000 daltons. In 1935, Pickels and Beams introduced new features of the centrifuge design that lead to its use as a preparative ultracentrifuge in cell fractioning. In 1938, Behrens employed differential centrifugation to separate nuclei and cytoplasm from liver cells. In the late 1940s early 1950s, Claude, Brachet, Hogeboom, and others had further developed this technique to fraction cell organelles (Alberts, 2002). **

Recent Research:
 * One of the most recent researches that involves centrifugation is “Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation regimes” which analyzes how centrifugation increases sperm concentration and cleans urine in contaminated samples (Nicholas, 2012). Another is the “ Enhanced detection of infectious salmon anaemia virus using a low-speed centrifugation technique in three fish cell lines” which a low-speed centrifugation is used to decrease time until CPE was observed (Molloy, 2012). **


 * Harding et al. (2012) used an ultracentrifuge in order to determine the hydrodynamic properties in solution of //Streptococcus pneumonia//e. This technique was necessary to find the sedimentation coefficient, molar mass distributions, solution conformations, and flexibilities. This information was used to create vaccines.**


 * Patel et al. (2010) used this technique in order to model the laminin gamma-1 short arm. The centrifuge was used to determine the sediment coefficient and was used to help determine the average molecular weight.**


 * Strom et al. (2010) used this technique to isolate LDL, HDL, and lipoprotein-deficient serum in order to study the effects of mutated genes on cholesterol levels.**

==== Centrifugation is a common research technique in many different areas of science. In an article by Yokoyama, centrifugation is a widely used technique to extract pore water from geological material. The centrifugation provided information on the solute distribution associated with reaction and transport occurring in rock pores. The scientists that preformed this study found that centrifugation was an effective method to examine the solute distribution in pores of various sizes. More data might have the potential to contribute to a greater understanding of water-rock reactions (Yokoyama, 2011). Centrifugation was also used as a method to concentrate protein solutions based on dialysis–freezing–centrifugation: Enzyme applications in an article by Virgen-Ortiez (2012). In this article, the authors were trying to access the problem of dilute proteins so they developed a process to concentrate enzymes. The centrifugation step in the process helped determine the proper concentration of proteins without effecting their enzymatic activity (Virgen-Ortiez, 2012). One last piece of research that used the centrifugation technique was in an article by dos Santos in 2010. In this article the authors tried to develop a new centrifugation procedure to help optimize the extraction of elements in marine invertebrates. The centrifugation was paired with sonication for 15 minutes and it was found to offer advantages such as precision and accuracy in determining the traces of elements in the invertebrates (dos Santos, 2010). ====

References:**

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Peter, W. (2002). //Molecular biology of the cell//. (4th ed., pp. 299-374). New York, New York: Garland Science.

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2008). //Molecular Biology of the Cell// (5th ed.). NY, NY: Garland Science.

Harding, S. E., Abdelhameed, A. S., Morris, G. A., Adams, G., Laloux, O., Cerney, L., & Bonnier, B. (2012, September). Solution properties of capsular polysaccharides from Streptococcus pneumoniae. //Carbohydrate Polymers//, //90//(1), 237-242.

Molloy, S. D., Thomas, E., Hoyt, K., & Bouchard, D. A. (2012). Enhanced detection of infectious salmon anaemia virus using a low-speed centrifugation technique in three fish cell lines. //Journal of Fish Diseases//, doi: 10.1111/j.1365-2761.2012.01412.x

Nicholas, M., Alvarez, M., ANel, E., Martinez, F., Borragan, S., Martinez-Pastor, F., de Paz, P.& Anel, L. (2012). Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation. //European Journal of Wildlife Research//, //58//(1), 77-84. doi: 10.1007/s10344-011-0544-8

Patel, T. R., Morris, G. A., Zwolanek, D., Keene, D. R., Li, J., Harding, S. E., & Koch, M. (2010, September). Nano-structure of the laminin y-1 short arm reveals an extended and curved multidomain assembly. //Matrix Biology//, //29//(7), 565-572.

Strom, T. B., Holla, O. L., Cameron, J., Berge, K. E., & Leren, T. P. (2010, February). Loss-of-function mutation R46L in the PCSK9 gene has little impact on the levels of total serum cholesterol in familial hypercholesterolemia heterozygotes. //Clinica Chemica Acta//, //411//(3-4).

==== dos Santos, W. (2010, July). Optimization of a centrifugation and ultrasound-assisted procedure for the determination of trace and major elements in marine invertebrates by ICP OES. //Microchemical Journal//, //95//(2). Retrieved October 10, 2012, from ScienceDirect. ==== ==== Virgen-Ortiez, J. (2012, July). Method to concentrate protein solutions based on dialysis–freezing–centrifugation. //Analytical Biochemistry//, //426//(1). Retrieved October 10, 2012, from ScienceDirect. ====