Biology Reference
In-Depth Information
3.2
Purifi cation of
the Cytosolic
Fraction from
Arabidopsis
Seedlings
All steps are performed at 4 °C except the infi ltration and incuba-
tion in digestion medium.
1. Protoplast isolation
(a) Harvest approximately 20 g of seedlings in the morning
before the light period starts (
see
Note 11
).
(b) In a beaker, add to the seedlings 30 mL Digestion Medium
and vacuum infi ltrate the plant material for 30 min (
see
Note 12
). Incubate for an additional 2.5 h in the
dark
. As
explained in
Note 5
, protoplast generation may vary with
the plant origin and its development. So, we optimized
this step using a ratio of 1:1.5 or 1:2 seedlings fresh weight
(FW) to Digestion Medium.
(c) Filter the mixture through two layers of Miracloth pre-
soaked with Wash Buffer. Collect the fi ltrate if Digestion
Medium is to be reused (
see
Note 13
).
(d) In a beaker, release the protoplast from undigested tissue
by resuspension in 60-90 mL Wash Buffer. The volume of
Wash Buffer is two to three times higher than the volume
of Digestion Medium used in
step 1b
. Release of proto-
plast is achieved by swirling the mixture on ice for several
minutes. Collect the dark-green eluent in a glass beaker
on ice. All subsequent steps up to
step 1i
must be per-
formed at 4 °C.
(e) Split extract into 30 mL Corex tubes, centrifuge for 5 min
at 500 ×
g
in a fi xed angle or swing out rotor and discard
the supernatant.
(f) Repeat Wash
steps 1d
and
1e
(
see
Note 14
).
(g) Resuspend pellets in 15 mL Flotation Medium I (FMI).
Then, overlay 7.5 mL of FMII followed by 3 mL FMIII
(
see
Note 15
).
(h) Centrifuge the gradient at 250 ×
g
for 5 min at 4 °C in a
swing-out
rotor (
brake off
).
(i) Recover the intact protoplasts from the FMIII/FMII
interface (Fig.
3
) and continue with homogenization.
Keep the pellet for chloroplast purifi cation (
see
step 4
).
2. Protoplast homogenization
(a) Disrupt the intact protoplasts using three to fi ve strokes of
a prechilled Potter-Elvehjem homogenizer at 4 °C, prefer-
ably in a cold room (
see
Note 16
).
(b) Transfer the solution containing the broken protoplasts to
a 15 mL Corex tube. Using a long-neck glass Pasteur
pipette, insert a 500
L cushion of 85 % (v/v) Percoll at
the bottom of the tube followed by a 1 mL layer of FMIII
on top of the protoplast homogenate (
see
Note 17
).
μ
