Biology Reference
In-Depth Information
18. Potter homogenizer, Wheaton USA.
19. Mortar and pestle.
20. Cold-room (4 °C).
21. Beakers, measuring cylinders and funnel.
22. Vacuum pump.
3
Methods
1. Sow approximately 3-5 seeds of
Arabidopsis thaliana
Col-0 in
each pot (15 pots per tray, ca. 1 tray, i.e., approx. 60 plants per
isolation) fi lled up with commercial soil and perlite.
2. Fill the tray ca. 1 cm with water and let the pots soak up the
water overnight. Discard excess water.
3. Keep the tray(s) in the cold-room for stratifi cation for 2 days.
4. Transfer the Arabidopsis tray(s) to a plant growth room main-
tained at ~22 °C and light intensity of 100 ~ 150
3.1 Growth of
Arabidopsis Plants
μ
mol/m
2
/s
in a 16/8 h cycle (long-day).
5. Cover the tray with a plastic dome in the fi rst week until ger-
mination to maintain high humidity.
6. Treat the plants with Hoagland nutrient solution [
15
] once a
week, if required.
7. Healthy
Arabidopsis
plants (4-5 weeks old, 60 g fresh weight
(FW) are generally required for high leaf peroxisome yield and
purity).
3.2 Isolation of Leaf
Peroxisomes
1. Harvest the plants in the end of the dark or (preferentially)
after an extended dark period (
see
Note 11
).
2. Cut the rosette plants at the top of the root and weigh 60-70 g
fresh weight (FW) for a single isolation. Wash the rosette plants
to remove all the dirt associated with them. Transfer the leaves
to a plastic bag and store between ice crushes until use (gener-
ally >2 h) (
see
Note 11
).
3. The isolation procedure should be started in a cold-room
(4 °C) or at least on ice using precooled solutions, equipment
(e.g., mortar), and centrifuges.
4. Transfer the Arabidopsis leaves from one plastic bag into the
precooled mortar and add 120 mL of grinding buffer (GB).
Grind the leaves with a pestle for ca. 5-10 min until the buffer
is dark green and individual leaves are no longer visible. For
small mortars it might be advantageous to split the leaves into
two equal portions (2× ca. 30 g FW) and grind each with
60 mL GB to increase the grinding effi ciency.
