Biology Reference
In-Depth Information
3
Methods
3.1 Protoplast
Preparation of
Arabidopsis Cell
Suspension Cultures
1. Maintain growing Arabidopsis cells in 100 mL aliquots, and
subculture weekly at 1:10 ratio. Filter 7-day-old cells from
approximately ten fl asks through Miracloth and fi rmly squeeze
out the remaining culture medium. Weigh out 60-80 g cells if
possible; otherwise work with no less than 30 g cells (
see
Note 5
).
2. For 80 g cells (fresh weight), use 800 mL of digestion buffer
(
see
Note 6
). Divide into equal volumes and add 0.4 % (w/v)
cellulase and 0.05 % (w/v) pectolyase to 400 mL of digestion
buffer in a 4 L wide-bottomed, conical fl ask. Add cells and
ensure that they are adequately suspended in the buffer. Place
on an orbital shaker and rotate slowly in the dark (wrapped in
foil) for 3 h (
see
Note 7
).
3. Protoplasts are harvested by centrifugation at 800 ×
g
for 5 min
(rotor with capacity for 250-500 mL), gently resuspended in
around 150 mL of the remaining digestion buffer (400 mL, no
enzymes), and centrifuged at 800 ×
g
and buffer discarded.
Repeating this step two more times ensures complete removal
of cellulase and pectolyase from the protoplasts (
see
Note 8
).
4. After the fi nal wash in digestion buffer (no enzymes), resus-
pend the pellet/protoplasts in homogenization buffer using a
minimum ratio of 1:1 (w/v) using the original fresh weight of
cells to buffer volume.
1. Working at 4 °C, homogenize the protoplasts in homogeniza-
tion buffer using 4-5 strokes at even pressure with a Potter-
Elvehjem homogenizer (
see
Note 9
).
2. Compare pre- and post-homogenized protoplasts under a light
microscope to ensure adequate disruption.
3. Centrifuge homogenate at 5,000 ×
g
for 15 min (rotor with
capacity for 50 mL samples).
4. Transfer the supernatant to an appropriately sized, chilled beaker;
slowly add 500 mM KCl drop-wise whilst gently agitating the
homogenate, until a fi nal concentration of 50 mM KCl is
reached; and then incubate at 4 °C for 5 min.
3.2 Homogenization
of Protoplasts and
Pre-FFE Enrichment
5. Add 5 mL of gradient buffer 1 to the required number of
40 mL ultracentrifuge tubes. Using a plastic Pasteur pipette,
gently layer the homogenate onto the cushion of gradient
buffer 1 until all tubes are at least two-thirds full (
see
Note 10
).
6. If collecting intact Golgi cisternae use gradient 1 and
ultracentrifuge for 1 h at 60,000 ×
g
. If collecting Golgi and
other secretory membranes use gradient 2 and ultracentrifuge
at 100,000 ×
g
(
see
Notes 11
).
