Biology Reference
In-Depth Information
Harvest the aerial part of plants
Homogenize in homogenizing medium
Centrifuge at 10,000 ×g for 15 min
Supernatant
Pellet
Centrifugeat 231,000 × g for 50 min
Pellet
Supernatant
Suspend with MS-suspension medium
and centrifuge at 231,000 × g for 50 min
Pellet
Supernatant
Suspend in MS-suspension medium,
put into two-phase partition medium
and centrifuge at 650 × g for 5 min
Lower phase
Upper phase
Put on a newly prepared lower phase
and centrifuge at 650 × g for 5 min
Lower phase
Upper phase
Put on a newly prepared lower phase
and centrifuge at 650 × g for 5 min
Lower phase
Upper phase
Suspend in PM-suspension medium
and centrifugeat 231,000 × g for 50 min
Pellet
Supernatant
Suspend in PM-suspension medium
and centrifuge at 231,000 × g for 35 min
Pellet
Supernatant
Suspend in PM-suspension medium
and store at −80°C
Fig.
1
Schematic outline of the plasma membrane preparation. All steps from harvesting leaves to suspending
the purifi ed plasma membrane fractions were described
and wash with 500 mL of chilled water. Wash twice. Drain on
a paper towel and put on crushed ice.
2. Cut into small pieces with razor blades. Immediately, put into
four volumes of chilled homogenizing medium and mix well
with a spatula. The homogenizing medium containing the
samples should be again cooled on crushed ice (
see
Note 12
).
3. Homogenize with a chilled Polytron generator (PT10SK,
Kinematica Inc., Lucerne, Switzerland) until the samples are
broken down into tiny pieces (speed 6 for 60-90 s). Filter
the homogenates through four layers of gauze and squeeze
tightly. Put the fi ltrates into 40 mL centrifuge tubes and
balance them in pairs. Centrifuge at 10,000 ×
g
for 15 min
with a chilled rotor to remove debris and heavy membrane
fractions (
see
Note 13
).
