Biology Reference
In-Depth Information
3
Methods
Although the extraction method outlined here is for chloroplasts,
it can also be used for any other type of plastid. All of the proce-
dures are performed at 4 °C.
3.1
Tissue Disruption
1. Wash 5-10 g of plant tissues (
see
Note 10
) with distilled water
and immediately put those on ice (
see
Notes 11
-
13
).
2. Add 30 mL of precooled (4 °C) plastid isolation buffer to a
stainless steel bath that is placed on ice. Cut the tissues into
small sizes (about 3 mm) using a razor or a dissecting scissors
washed with acetone or ethanol (
see
Note 14
).
3. Wrap the tissues in two layers of Miracloth and crush them
using a mortar and pestle.
3.2
Filtration
1. Filter the homogenate through two layers of Miracloth and
collect the fi ltrate.
2. Homogenize the remaining tissue in the Miracloth again and
collect the fi ltrate.
3. Pool the fi rst and second fi ltrates together and fi lter through
four layers of Miracloth.
4. Centrifuge the fi ltrate at 200 ×
g
for 4 min at 4 °C to remove
the nuclei and cellular debris. Collect the supernatant.
5. Centrifuge the supernatant at 2,000-2,500 ×
g
for 4 min at
4 °C to precipitate the chloroplasts (
see
Note 15
). Discard the
supernatant.
6. Resuspend the pellet containing chloroplasts in 3 mL of plastid
isolation buffer. Vortex gently (
see
Note 16
).
3.3 Percoll Density
Gradient
Centrifugation and
Intact Plastid Isolation
1. Layer the chloroplast suspension slowly onto a discontinuous
Percoll density gradient solution containing 1 mL of 80 % and
3 mL of 40 % Percoll (
see
Note 17
).
2. Centrifuge at 4,000 ×
g
for 10 min at 4 °C using a swing bucket
rotor (P40ST, Hitachi).
3. After centrifugation, intact chloroplasts will be on the interface
of the 40 and 80 % Percoll (Fig.
1
). The band on the upper
layer will contain broken chloroplasts and thylakoid mem-
branes. Use a Pasteur pipette to collect the fraction that con-
tains intact chloroplasts.
4. Add fi ve times the volume of plastid isolation buffer to the col-
lected fraction and wash gently.
5. Centrifuge at 4,000 ×
g
for 10 min at 4 °C. Discard the
supernatant.
