Biology Reference
In-Depth Information
11. Miracloth (Merck KGaA, Germany).
12. Pasteur pipettes (glass) with long neck.
13. 5 kDa Ultrafree centrifugal fi lter devices (EMD Millipore,
MA, USA) for protein concentration.
14. Preparative centrifuge with swing out rotors capable of pro-
cessing 6 × 30 mL samples at up to 800 ×
g
and 4 × 150 mL
samples at up to 3,000 ×
g
.
15. Ultracentrifuge with fi xed angle rotor capable of processing
4 × 10 mL samples at 100,000 ×
g
.
16. Vacuum pump, such as Vacuubrand model MZ 2C or similar
(Wertheim, Germany), capable of vacuum 9.0 mbar and maxi-
mal pumping speed of 1.7/2.0 m
3
/h.
17. Corex centrifuge tubes, 15 and 30 mL capacity.
18. Collection of plant organelle marker antibodies (Agrisera AB,
Vännäs, Sweden).
3
Methods
Carry out all procedures at room temperature unless otherwise
specifi ed.
3.1 Purifi cation
of the Cytosolic
Fraction from Plant
Cell Cultures
1. Protoplast production
(a) Collect cells and remove cell culture medium by fi ltering
through two pieces of Miracloth.
(b) Use a ratio of 1:5 plant cells fresh weight (FW) to Enzyme
Buffer (i.e., 10 g cells to 50 mL buffer) to generate proto-
plasts from
Arabidopsis
cells culture (
see
Note 5
).
(c) Resuspend fi ltered cells in Enzyme Buffer and incubate in
a wide-base conical fl ask for 3 h with gentle orbital rota-
tion (~85 rpm) in the dark (
see
Note 6
).
(d) Harvest protoplasts by centrifuging at 800 ×
g
for 10 min
at 4 °C. Gently resuspend the pellet (protoplasts) with
Wash Buffer using a 1:5 ratio of cells to buffer. Centrifuge
at 800 ×
g
for 10 min at 4 °C and repeat for a total of two
washes with Wash Buffer. Proceed with homogenization
(
see
Note 7
).
2. Protoplast homogenization
(a) Resuspend protoplasts in Homogenization Buffer and
disrupt them with about fi ve strokes of a Potter-Elvehjem
homogenizer at 4 °C. Use a ratio of 1:2 cells FW to
Homogenization Buffer. This step is preferably done in a
cold room (
see
Note 8
).
