Biomedical Engineering Reference
In-Depth Information
Table 5.3.
Buoyant density of mitochondria and DNA from
Micrococcus
luteus
(G+C
71
%), dependent on the applied density gradient medium
Medium
Mitochondria
DNA
/
cm
3
)
(g
Metrizamide
1.20 - 1.25
1.12 - 1.17
Sucrose
1.19
Percoll/sucrose
1.09 - 1.11
Ficoll
1.14
Cesium chloride
1.73
Cesium sulfate
1.45
Cesium trifluoracetate
1.63
Potassium iodide
1.51
Sodium iodide
1.55
A
0.9% NaCl (w/v) in pure water (saline)
Solutions/Reagents
B5mM MgCl
2
,20mM Tr i s
·
HCl, pH 7.4
C . 5M sucrose in Soln. B
D . 4M sucrose in Soln. B
E
1% Triton X-100 (w/v) in Soln. C
Cool freshly prepared rat liver in ice-cold saline and mince using
scissors. Decant the liquid and add 9 vol. (with respect to tissue
weight) of ice-cold Soln. B. Homogenize the tissue in a pre-cooled
glass-Teflon Potter-Elvhjem homogenizer with about 2000 rpm
and eight to ten strokes. Filter the homogenate through several
layers of cheesecloth.
Fill lockable centrifuge tubes to 40% with ice-cold Soln. D and
cover with the same volume of homogenate. Spin in a refrigerated
centrifuge with 1000
g
max
at 4
◦
C for 10 min. Discard the super-
natant and suspend the pellet in the original volume of Soln. E.
Spin and wash again as described.
Suspend the washed nuclei in 2 ml of ice-cold Soln. C per rat
liver.
×
References
Widnell CC, Tada JR (1964) Biochem J 92:331
5.3.2 Sucrose Gradient Centrifugation:
Preparation of Surface Membranes (Sarcolemma, SL)
of Heart Muscle Cells
This protocol illustrates the combination of differential and density
gradient centrifugation, using a high-capacity low-speed centrifuge
as well as an ultracentrifuge.
