gradient assembled in an ultracentrifuge tube (
see
Note 10
and
Subheading
2.2
item 11
, Fig.
2
). Load 5 mL of 36 % (w/w)
sucrose solution on the second density gradient needed as a
counterbalance for the leaf peroxisome gradient during
ultracentrifugation.
16. Place the two gradients in two precooled SW41 Ti buckets.
Carefully balance the fi lled and the empty SW41 Ti buckets
and spin for 40 min to 2 h at 80,000 ×
g
(
see
Note 16
).
17. After centrifugation a white band of leaf peroxisomes should
be visible at the interface of the 50.5 % (w/w) and 55.2 %
(w/w) sucrose density gradient fractions (Fig.
2
). Suck off the
upper fractions with a Pasteur glass pipette attached to a vac-
uum pump. Harvest the peroxisome band in a volume of
approx. 1 mL and adjust the volume in a 1.5-mL Eppendorf
tube to 1.5 mL using 36 % (w/w) sucrose solution or TE buf-
fer only (
see
Note 17
).
18. Add the following protease inhibitors at the following fi nal
concentrations: PMSF (1 mM), Benzamidine (2 mM),
ɛ
-Aminocaproic acid (2 mM), Aprotinin (1
g/mL). Prepare a second
Eppendorf tube as blank for protein determination containing
approx. the same sucrose concentration in TE buffer (ca. 45 %
w/w) and the same fi nal concentrations of the six protease
inhibitors.
19. Mix well and prepare a few 50-
L aliquots for analysis of pro-
tein concentration and purity using enzymatic assays such as
HPR activity and immunoblotting (Table
2
) and freeze all
samples at −20 °C (
see
Note 18
).
20. To concentrate proteins for subsequent proteome studies we
recommend chloroform/methanol precipitation [
16
].
1. If two Sorvall centrifuges are available, one person can carry out
two leaf peroxisome isolations in ca. 4-5 h, and one to two
persons can carry out four to six leaf peroxisome isolations per
day. The average yield of one isolation is ca. 120
g protein [
7
].
2. All solutions are generally prepared on the previous day and
stored in the refrigerator. The pH of solutions is adjusted after
precooling to ca. 10 °C.
3. Stock solutions such as 1.0 M Tricine, 100 mM EDTA, 1.0 M
KCl, and 1.0 M MgCl
2
are recommended to facilitate buffer
preparation.
4. Appropriate stock solutions such as 100 mM PMSF (in
MeOH), 200 mM Benzamidine (in H
2
O, freshly prepared),
