Biology Reference
In-Depth Information
(a)
(b)
high
high
low
low
top
bottom
top
bottom
TUBE
DISCONTINUOUS
GRADIENT
TUBE
CONTINUOUS
GRADIENT
FIGURE 12.7
Density Gradient Centrifugation density profiles: (a) Discontinuous (or step) gradient and;
(b) Continuous gradient.
fashion, it is possible to tailor a specific gradient fraction to match the density property of the
membrane of interest and maximize its yield.
Figure 12.8
shows a discontinuous gradient of
various membrane fractions isolated from rat brain homogenate
[27]
. Densities of the five
fractions were determined by the amount of Percoll (see below) in each fraction (0%, 3%,
10%, 15%, and 23%).
A continuous gradient displays a linear decrease in solution density from the bottom to
thetopofthecentrifugetube(
Figure 12.7
b). Gradients are generated by either a 'gradient
maker' or can be self-generating during the centrifugation.
Figure 12.9
demonstrates two
Rat brain homogenate
Centrifuge
10 min, 1,000
g
P1: Pellet
S1: Supernatant
Centrifuge
5 min, 31,000
g
Percoll
0%
F1: Membranes
3%
F2: Myelin and
membranes
10%
F3: Synaptosomes
and membranes
15%
F4: Synaptosomes
23%
F5: Mitochondria
FIGURE 12.8
Discontinuous (step) gradient of various membrane fractions isolated from rat brain homogenate.
Density of the 5 fractions was established by the amount of Percoll in each fraction (0%, 3%, 10%, 15%, and 23%)
[27]
.
