Biology Reference
In-Depth Information
7. Remove the lipids from the inner tube walls carefully with a
paper towel. Do not touch the pellet!
8. Resuspend the pellet in centrifugation buffer (
see
Note 18
).
Usually, 2 × 500
l are suffi cient. This is the microsomal or the
membrane fraction which contains all endomembranes (ER,
Golgi, mitochondria, vacuole) and the plasma membrane.
Determine the protein concentration of the fractions (e.g.,
Lowry assay compatible with DTT!) and store it at −20 °C
(1 month) or −80 °C (1 year).
μ
Although this isolation method is quite old, the big advantage is
the possibility to isolate vacuolar, ER, Golgi, mitochondrial, and
plasma membrane vesicles in one preparation step simultaneously
and from the same pool of pollen grains. Aqueous two-phase parti-
tioning may get organelle fractions of higher purity but can be opti-
mized just for the isolation of one membrane fraction. A preparative
free-fl ow electrophoresis system also allows the simultaneous
separation and isolation of different organelle vesicles but is
extremely expensive and labor intensive.
3.4 Isolation
of Organelle
Membranes on a
Discontinuous Sucrose
Density Gradient
1. Prepare the sucrose solutions the day before and store them
at 4 °C.
2. Prepare the sucrose step gradient in a high-speed centrifuga-
tion tube (36 ml) by carefully pipetting layers of sucrose solu-
tions. Start with the highest sucrose concentration: 4 ml of
45 % followed by 7 ml of 38 %, 34 %, 30 %, 4 ml of 25 %, and
fi nally 4 ml of 18 % sucrose solution (
see
Note 19
). Experienced
researches may add the sucrose solutions manually using a
normal pipette but sucrose layers should not be mixed!
3. Layer up to 1 ml MF on top of the sucrose gradient.
Homogenize the MF after it has been thawed up on ice with a
small glass homogenizer. Keep the MF cool.
4. Tare the centrifugation tubes carefully to at least 1 mg toler-
ance and centrifuge in a swing-out rotor at 100,000 ×
g
for 2 h
15 min at 4 °C (Sorvall rotor AH-629, ca. 28,000 rpm).
5. Collect the interphases (Fig.
4a
) with a Pasteur glass pipette
whose opening has been bended by 90°, into reaction tubes.
Keep interphases on ice.
6. Determine the sucrose concentration of the interphase frac-
tions using a refractometer.
7. Dilute the interphase fractions to at least 10 % sucrose and pel-
let the membranes by centrifugation at 145,000 ×
g
for 60 min
at 4 °C in a fi xed-angle rotor.
8. Discard supernatant and resuspend the pellet carefully in
centrifugation buffer (
see
Note 18
).
9. The fractions can be stored at −80 °C for up to 3 months
without severe loss of enzyme activities.
