Biology Reference
In-Depth Information
4. Transfer the supernatants into ultracentrifuge tubes by
decantation. Centrifuge at 231,000×
g
for 50 min with a
chilled ultracentrifuge rotor to precipitate the microsome frac-
tions. Discard supernatants by decantation.
5. Add an appropriate quantity of MS-suspension medium to
each tube and homogenize the pellets with a Tefl on-glass
homogenizer. Collect the microsomal suspensions with a large
Pasteur pipette into ultracentrifuge tubes. Balance ultracentri-
fuge tubes in pairs with MS-suspension medium.
6. Ultracentrifuge at 231,000 ×
g
for 50 min as described in
step
4
. Put 5 mL of MS-suspension medium in a Tefl on-glass
homogenizer and mark the water surface on the glass homog-
enizer as an indication of 5 mL volume. After centrifugation,
discard the supernatant with an aspirator.
7. Put 2 mL of MS-suspension medium and break up the precipi-
tated pellet with a glass rod. Transfer into a Tefl on-glass
homogenizer using a large Pasteur pipette. Put 2 mL of
MS-suspension medium into the tube and pipette up and
down to break up the remaining pellet. Transfer into a Tefl on-
glass homogenizer and add MS-suspension medium to 5 mL.
Homogenize well with an electric Tefl on-glass homogenizer
(moving up and down fi ve times) with cooling on ice
(
see
Note 14
).
8. Put all of the homogenate in a centrifuge tube containing two-
phase partition medium (tube A). Put 5 mL of MS-suspension
medium to other two-phase partition systems (tubes B and
C). Chill on crushed ice for 10 min. During this time, mix well
every 2 min.
9. Centrifuge tubes A and B at 650×
g
for 5 min in a chilled
rotor. Two phases should be observed to have settled in tubes
A and B. Discard the upper phase of tube B with a Pasteur
pipette and transfer the upper phase of tube A into tube B.
Chill on crushed ice for 10 min. During this time, mix well
every 2 min (
see
Note 15
).
10. Centrifuge tubes B and C at 650×
g
for 5 min in a chilled
rotor. Discard the upper phase of tube C with a Pasteur pipette
and transfer the upper phase of tube B into tube C. Balance
tube C with another centrifuge tube fi lled with water. Chill on
crushed ice for 10 min. During this time, mix well every 2 min
(
see
Note 15
).
11. Centrifuge at 650 ×
g
for 5 min and split the resultant upper
phase of tube C into two ultracentrifuge tubes. Fill up the
tubes with PM-suspension medium and balance them.
Ultracentrifuge at 231,000 ×
g
for 50 min, as described in
step
4
(
see
Note 15
).
