Biology Reference
In-Depth Information
(b) Centrifuge at 250 ×
g
for 5 min at 4 °C in a swing-out rotor.
(c) Remove as much of the supernatant as possible and gently
resuspend the pellet in a small volume (0.5-1 mL) of the
same buffer.
(d) Using a cut-off pipette tip, carefully load the resuspended
chloroplast mixture onto a 45/85 % (v/v) Percoll™
gradient (
see
Note 18
), and centrifuge at 3,000 ×
g
15 min
at 4 °C in a swing-out rotor (
brake off
).
(e) With a long-neck Pasteur pipette, collect intact chloro-
plasts from the 45/85 % (v/v) interface (
see
Note 19
)
(Fig.
3
) and transfer them in a new tube.
(f) Prior to centrifugation at 800 ×
g
for 15 min in a swing-
out rotor at 4 °C, wash with 10 volumes of Chloroplast
Buffer to dilute the Percoll™.
(g) Finally, resuspend the intact, clean chloroplasts in 500 mL
of Chloroplast Buffer, or 10 % (w/v) TCA in acetone if a
total protein extraction is required.
5. Purity Assessment
(a) Assess purity of both cytosolic and chloroplast fractions by
immunoblotting with commercially available antibodies
against plant organelle protein markers (e.g., mitochon-
dria, plastids, nuclei, the ER, plasma membranes, peroxi-
somes, and cytosol (Agrisera AB, Vännäs, Sweden))
(Fig.
2
).
4
Notes
1. It is critical to freshly dissolve the plant cell wall degrading
enzymes (cellulase and pectolyase) in enzyme buffer immedi-
ately prior addition to plant cells. The enzyme buffer solution
contains: mannitol (acting as osmoticum to maintain isotonic
conditions between the medium and plant cells), MES buf-
fer (to achieve optimal pH for
Arabidopsis
cell suspension)
and cellulase and pectolyase (a mix of enzymes that degrade
the cellulosic and pectic components of plant cell walls). Wash
Buffer is required for subsequent wash steps to remove
residual cellulase and pectolyase from the protoplasts.
2. The osmoticum maintains the integrity of organelle membranes.
The buffer counteracts acidifi cation from ruptured vacuoles,
EDTA chelates divalent metal ions required by phospholi-
pases and various proteases and the reductant DTT prevents
damage from oxidizing agents.
3. It is preferable to freshly prepare the Digestion Buffer.
However, Digestion Buffer can be used if stored at −20 °C
for up to 6 months.
