Technical information

Centrifuge Standard Operating Procedure 2
2
2
1
1
k
T
k
T

When using a specific rotor, you can determine how quickly a given particle
will pellet by using the sedimentation coefficient (S) for that particle. The S-
value is expressed in Svedberg units and a larger S-value indicates faster
sedimentation. The time taken to pellet a given particle can be determined by:
S
k
T

where T= pellet time in hours. k = pelleting efficiency of the rotor, and S =
sedimentation coefficient of the particle.
1.2.1 Centrifugation Methods
A variety of methods have been developed to enable different types of
separation using centrifugation, and these include differential centrifugation
and density gradient centrifugation.
Differential centrifugation separates particles based on size, and is typically
used for pelleting and partial purification of subcellular organelles and
macromolecules. Subcellular component purification is achieved by using a
series of successively higher g-force centrifugations followed by density
gradient separations.
A density gradient separation uses continuous or discontinuous layered gradient
media to separate subcellular organelles and macromolecules. There are two
types of density gradients: Rate zonal, which separates based on size or mass,
and isopycnic, which separates based on density. Rate zonal separations are
commonly applied to separate cellular organelles or proteins, and cannot be
run too long or all of the components will pellet at the bottom. During an
isopycnic separation, each particle sinks until it reaches a layer where the
density of the medium is the same as the density of the particle. In this case
the centrifugation run must be run long enough to ensure that all particles have
separated, and excessive run times will not have an adverse affect on the
separation. Cesium chloride separation of nucleic acids is an example of an
isopycnic separation.