Structural Biochemistry/Proteins/Zonal Centrifugation

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Zonal Centrifugation (Sedimentation Coefficient)[edit]

Another method in determining protein size is zonal centrifugation. Also known as band or gradient centrifugation, this technique relies on the concept of the sedimentation coefficient. The sedimentation coefficient is an equation that quantifies the rate of movement through a liquid medium through the formula:

s = m (1-vp)/f

where s = sedimentation coefficient, m = mass, v = partial specific volume, p = density of the medium, and f = frictional ratio. The unit of this equation are Svedberg units (S), which is equal to 10-13 s. A smaller S value generally means that a molecule will move more slowly in a centrifugal field, as opposed to a higher S value.

Some important conclusions that can be drawn from this equation include:

  1. Since the velocity of a particle depends on its mass, particles with higher mass will sediment faster than particles with less mass.
  2. Shape also determines the rate of sedimentation since it affects viscous drag. Therefore a more compact particle will have a smaller frictional coefficient than that of an elongated particle with the same mass. This means that more compact particles will sediment faster than elongated particles (same mass).
  3. The sedimentation velocity is dependent upon the density of the solution (p). Particles that have a vp value less than 1 will sink, while particles that have a vp value greater than 1 will float. Particles that have a vp value equal to 1 won't move.[1]

To use this technique, a density gradient is first created in a test tube (usually with sucrose) with the highest density at the bottom. The purpose of the density gradient is to prevent convective flow. A sample of proteins is then placed on top of the gradient and then centrifuged. The proteins separate accordingly to their sedimentation coefficient into bands which can then be collected by creating a hole at the bottom of the tube. [1]

Zonal Centrifugation

The diagram below illustrates a simplified version of this technique with DNA as a sample instead.


File:DNA sucrose gradient.jpg
Zonal Centrifugation

The benefit of using this technique is that it is very accurate and can be done without denaturing the protein.

References[edit]

1. Berg, Jeremy Mark, John L. Tymoczko, and Lubert Stryer.Biochemistry. 6th. New York: W H Freeman & Co, 2006. Print.

2. "Centrifugation: Buoyant Density Centrifugation." Cell Fractionation. N.p., n.d. Web. 16 Nov. 2012. <http://www.freewebs.com/ltaing/centrifugation.htm>.

3.Berg, Jeremy, Tymoczko J., Stryer, L.(2012). Zonal Centrifugation.Biochemistry(7th Edition). W.H. Freeman and Company.