Biology Reference
In-Depth Information
2.2 Components
for Leaf Peroxisome
Isolation
The components and solutions given below are suffi cient for one
isolation of Arabidopsis leaf peroxisomes, which requires eight
Percoll density gradients (
see
Notes 1
-
3
).
1. Grinding Buffer (GB, 120 mL): 170 mM Tricine, pH 7.5,
1.0 M sucrose, 2 mM EDTA, 1 % (w/v) BSA, 10 mM KCl,
1 mM MgCl
2
, 0.5 % (w/v) PVP-40, 5 mM DTT. DTT should
be added freshly on the preparation day. Prior to use, the GB
is supplemented with protease inhibitors (fi nal concentrations:
0.1 mM PMSF, 0.2 mM Benzamidine, 0.2 mM
ɛ
-Aminocaproic
acid) (
see
Note 4
).
2. TE buffer: 20 mM Tricine-KOH, pH 7.5, 1 mM EDTA
(
see
Note 3
).
3. 15 % (v/v) Percoll solution (30 mL): 4.5 mL Percoll, 750 mM
sucrose, 0.2 % (w/v) BSA in 150 ml TE buffer (
see
Notes 3
,
5
and
6
). For one leaf peroxisome isolation 24 mL (8 × 3 mL) is
required.
4. 38 % (v/v) Percoll solution (100 mL): 38 mL Percoll 750 mM
sucrose, 0.2 % BSA in TE buffer (
see
Notes 3
,
6
and
8
). For
one leaf peroxisome isolation 88 mL (8 × 11 mL) is required.
5. 36 % (w/w) sucrose in TE buffer (ca. 150 mL, ca. 1.22 M
sucrose): ca. 62.6 g sucrose in TE buffer. The exact concentra-
tion of 36 % (w/w) is adjusted using a refractometer (
see
Notes
3
,
7
and
8
).
6. 38 % (v/v) Percoll: 36 % (w/w) sucrose = 2:1 (ca. 21 mL): Mix
two volumes of 38 % (v/v) Percoll (e.g., 14 mL) with one vol-
ume of 36 % (w/w) sucrose (e.g., 7 mL). For one leaf peroxi-
some isolation 16 mL (8 × 2 mL) is required.
7. 38 % (v/v) Percoll: 36 % (w/w) sucrose = 1:2 (ca. 21 mL): Mix
one volume of 38 % (v/v) Percoll (e.g., 7 mL) with two vol-
umes of 36 % (w/w) sucrose (e.g., 14 mL). For one leaf per-
oxisome isolation 16 mL (8 × 2 mL) is required.
8. 60 % (w/w) sucrose in TE buffer (ca. 50 mL ca. 2.26 M):
38.6 g sucrose in 50 ml TE buffer (
see
Notes 3
and
7
).
9. Percoll density gradient (1st gradient, Fig.
1
): Assemble eight
discontinuous Percoll density gradients in SS34 tubes in the
following manner (
see
Note 9
).
(a) Fill 11 mL 38 % (v/v) Percoll solution into an empty SS34
tube.
(b) Underlay with 2 mL 38 % (v/v) Percoll: 36 % (w/w)
sucrose = 2:1.
(c) Underlay with 2 mL 38 % (v/v) Percoll: 36 % (w/w)
sucrose = 1:2.
(d) Underlay with 3 mL 36 % (w/w) sucrose in TE buffer.
(e) Overlay with 3 mL 15 % (v/v) Percoll solution.
