Biology Reference
In-Depth Information
heavy particle A and light particle B. The initial centrifugation pellets ~95% of A, but also
pellets ~20% of B. If the pellet is separated, re-suspended in fresh media and re-centrifuged
at the same speed, ~85% of the initial amount of A is found in the pellet, but only ~1% of B.
Therefore, differential centrifugation can only produce enriched fractions that may be further
purified by repeated centrifugations. However, each centrifugation results in a significant
loss of the target membrane. Final purification is often accomplished through density
gradient centrifugation.
Density Gradient Centrifugation
Density gradient centrifugation is based on generating a centrifugation media that
is densest at the bottom of the centrifuge tube and least dense at the top of the tube.
The density gradient is used to separate particles on the basis of size and density.
How fast something moves in a centrifugal field is defined by the sedimentation
coefficient,
s
.
r
2
s ¼ 2
ðr
p
r
m
Þ=9h
where:
s
¼
sedimentation coefficient
r
radius of the particle
r
p
¼
¼
density of the particle
r
m
¼
density of the media
h¼
viscosity of the media
From this equation it can be deduced that: bigger particles centrifuge faster than smaller
particles; centrifugation rate is increased with increasing difference between density of the
particle and density of the media; and centrifugation rate is slowed by an increase in media
viscosity. Normally the sedimentation coefficient
s
has units of 10
13
. To avoid this cumber-
some unit, the sedimentation coefficient is multiplied by 10
13
generating the more commonly
used term,
S
, the Svedberg Unit.'
S ¼ s 10
13
Density gradients are of two basic types, discontinuous (or step,
Figure 12.7
a and contin-
uous
Figure 12.7
b). In most cases the cell homogenate is placed on top of the medium in the
centrifuge tube and the centrifugation is started. The cell fractions will migrate down the
density gradient until
r
p
¼ r
m
and
s
drops to 0. At this point the centrifugation is stopped
and the now separated cell fractions are individually isolated.
A discontinuous (or step) gradient is made by layering solutions of decreasing density on
top of one another. The densest solution is placed at the bottom of the tube with successively
lower density solutions on top (
Figure 12.7
a). The solutions are carefully layered by use of
a pipette or syringe. To prevent unwanted mixing of the solutions during gradient formation,
the centrifuge tube is tilted ~20 degrees from perpendicular as the solutions are layered. This
method is inexpensive and requires no special 'gradient making' equipment. Since the solu-
tion volumes can vary from layer to layer and the densities need not decrease in a linear
