Biomedical Engineering Reference
In-Depth Information
Table 5.1.
Substances used in density gradient centrifugation
Ionic
Non-ionic
Cesium chloride
Deuterium oxide (“heavy water”)
Cesium sulfate
Sucrose
Cesium trifluoracetate
Glycerol
Potassium iodide
Ficoll
Sodium iodide
Percoll
3,5-Diacetamido-2,4,6-
2-(3-Acetamido-5-N-methylacetamido-
triiodobenzoic acid
2,4,6-triiodobenzamido)-2-desoxy-d-
(metrizoic acid)
glucose (Metrizamide)
Nycodenz
Iodixanol (OptiPrep)
Table 5.2.
Density, viscosity, and concentration of aqueous density gra-
dient solutions at 20 °C
/
cm
3
)
Medium
Density (g
Viscosity
Conc. (w/w)
(N
×
s
×
m
−2
)
Cesium chloride
1.229
25
Sucrose
1.104
2.5
25
Glycerol
1.056
2
24
25
a
Metrizamide
1.134
1.9
24
a
Ficoll
1.09
37
Percoll
1.13
10
23
a
%w/v
that solubility decreases with decreasing temperature, which means
that at room temperature, a substance is dissolved, whereas at 4
◦
C,
crystals may be formed which could damage the centrifuge tube at
high centrifugal force.
As a consequence of high osmolarity, macromolecules or or-
ganelles are dehydrated resulting in altered sedimentation behav-
ior; thus, different buoyant densities for nucleic acids and mito-
chondria are observed in different density gradient media, as illus-
trated in Table 5.3. Data of some density gradient media are given
in Table 5.4.
5.3.1 Pre-formed Discontinuous Gradient Centrifugation:
Isolation of Liver Cell Nuclei
The preparation of cell nuclei from rat liver is an example of the ap-
plication of a pre-formed stepwise gradient: The tissue homogenate
is placed on top of a cushion of concentrated sucrose solution, and
only the dense nuclei cross this cushion during centrifugation.
