Biomedical Engineering Reference
In-Depth Information
increasing centrifugal forces, resulting in several pellets containing
particles with decreasing sedimentation rate.
As an example for differential centrifugation, this protocol gives
the preparation of membranes of the sarcoplasmic (intracellular)
reticulum (SR) of heart muscle.
A 0mM NaHCO
3
,5mM NaN
3
,0.1mM PMSF
1
,pH7.0
Solutions/Reagents
B .6M KCl, 20 mM Tris-maleate, 0.1 mM PMSF, pH 6.8
C . 5M sucrose, 5 mM histidine
·
HCl, pH 6.8
Carry out all operations at 2 - 8
◦
C.
Smash deeply frozen pieces of heart (easily done with a hammer,
ifthetissueiscooledbyliquidnitrogen).Thawportionsof20g
with 100 ml of cold Soln. A and homogenize in a Warring blender
twice at maximal speed for 30 s each. Transfer the homogenate into
a glass cylinder and homogenize with a Polytron rotating-blades
homogenizer at 40% of maximal power, three times for 10 s each.
Remove tissue fibers from the homogenizer between each run.
Combine the homogenates.
Spin the homogenate in a refrigerated centrifuge at 3000
gfor
20 min. Filter the supernatant through glass wool to remove fat and
tissue particles and centrifuge in a pre-cooled fixed-angle rotor at
8700
×
×
g
max
for 20 min. Repeat filtration and centrifugation.
Centrifuge the combined supernatants with 100 000
×
gfor
45 min (e.g., Beckman Coulter Type 45 Ti rotor; 35 000 rpm).
Discard the supernatant and resuspend the precipitated SR vesi-
cles in total 1 - 2 ml of Soln. C, freeze quickly in liquid nitrogen and
store aliquots at −70
◦
C.
References
Harigaya S, Schwartz A (1969) Circ Res 25:781
5.3 Density Gradient Centrifugation
Density gradients may be divided into three types: preformed dis-
continuous gradients (e.g., Protocol 5.3.1), preformed continuous
gradients (e.g., Protocol 5.3.2) or self-generating gradients (e.g.,
Protocol 5.3.3). Materials used for density gradients are classified
as ionic and non-ionic media (Table 5.1).
Gradients made by a distinct substance are not only charac-
terized by the density, but also by viscosity and ionic strength
and osmolarity (Table 5.2). Since gradient solutions of high den-
sity mostly are concentrated solutions, it should be kept in mind
1
Add 0.1 M PMSF stock in ethanol, submerging the pipette immediately
before use, and mix thoroughly, because PMSF is rapidly inactivated by
hydrolysis.
