Biology Reference
In-Depth Information
a
Protoplasts
c
Cytosolic
fraction
Intact
protoplasts
FMIII
(kDa)
MW
Chloroplast
100
75
Disruption
+
Spin
FMII
50
b
37
Broken
Chloroplast
25
20
15
10
45%
Chloroplast
Intact
Chloroplast
85%
SDS-PAGE
Fig. 3
Method for simultaneous isolation of cytosolic and chloroplast fractions from
Arabidopsis
seedlings. (
a
)
Protoplasts from 16-day-old seedlings are produced by digestion of cell walls using a buffer containing 1.5 %
(w/v) cellulase, 0.4 % (w/v) macerozyme, 0.5 M sucrose. Intact protoplasts are then isolated on a density
gradient between FMII (0.4 M sucrose, 0.1 M sorbitol) and FMIII (0.5 M sorbitol). Intact protoplasts are disrupted
with six strokes of a Potter-Elvehjem homogenizer on ice. Broken protoplasts are then centrifuged in the
presence of a bottom cushion of 85 % (v/v) Percoll and top layer of FMIII to clear them from contaminating
chloroplasts and unbroken protoplasts. (
b
) The chloroplast rich fraction is resuspended in buffer containing
0.33 M sorbitol, placed on top of a 45/85 % (v/v) Percoll gradient and centrifuged. Intact chloroplasts are recov-
ered in the 45/85 % (v/v) Percoll interface. (
c
) Total protein from seedlings (Total), intact protoplasts, chloro-
plasts, and cytosol is extracted in the presence of 10 % (w/v) TCA in acetone, separated by SDS-PAGE (4-12 %)
and visualized after staining. Representative protein profi les are shown for each isolated fraction, with enrich-
ment of RuBisCO large subunit (
arrow
) in chloroplast samples and its depletion in cytosolic fraction
3. Isolation of the cytosolic fraction
(a) Centrifuge at 2,000 ×
g
for 10 min at 4 °C in a swing-out
rotor (
brake off
). Unbroken protoplasts are in the FMIII/
sample interface, while chloroplasts should sit on top of
the 85 % (v/v) Percoll™ cushion (Fig.
3
).
(b) Very carefully, recover the middle phase containing the
cytosolic fraction using a clean long-neck glass Pasteur
pipette, and centrifuge this fraction at 100,000 ×
g
for
30 min at 4 °C. This will remove any contaminating
organelles and membranes.
(c) Concentrate the supernatant (cytosolic fraction) using a
5 kDa Ultrafree centrifugal fi lter device (Millipore) (
see
Note 10
).
4. Isolation of chloroplasts
(a) Using a long-neck Pasteur pipette, recover the material on
top of the 85 % (v/v) Percoll cushion from
step 3a
and
gently resuspend in 10 mL of Chloroplast Buffer.
