Biology Reference
In-Depth Information
1. 12 min at 13,000xg
2. 20 min at 27,000xg
ca. 19 mL
supernatant S1
3 mL 15% (v/v) Percoll
11 mL 38% (v/v) Percoll
2 mL 38% (v/v) Percoll: 36% (w/w) sucrose = 2:1
2 mL 38% (v/v) Percoll: 36% (w/w) sucrose = 1:2
3 mL 36% (w/w) sucrose
Leaf
peroxisomes
Fig.
1
Leaf peroxisome enrichment from Arabidopsis by Percoll density gradient centrifugation (fi rst density
gradient). The gradient consists of two different Percoll fractions (15 and 38 % (v/v), in 0.75 M sucrose,
top
), a
36 % (w/w) sucrose cushion (
bottom
) and two intermediate 2-mL fractions comprising mixtures of 38 % (v/v)
Percoll and 36 % (w/w) sucrose solution at ratios of 2:1 and 1:2. After centrifugation (12 min at 13,000 ×
g
and
20 min at 27,000 ×
g
) the leaf peroxisomes are located at the
bottom
of the centrifugation tube. The fi gure has
been adapted and reprinted from Reumann et al. [
7
] Suppl. Figure 1
10. Prepare the solutions for the sucrose density gradient in the
described manner (Table
1
) using the 60 % (w/w) and 36 %
(w/w) stock solutions.
11. Sucrose density gradient (2nd gradient): Assemble two discon-
tinuous sucrose density gradients in two ultracentrifuge tubes
(e.g., Beckmann SW41 Ti, 13 mL) in the following manner
(Fig.
2
,
see
Note 10
):
(a) Fill 1.0 mL 55.2 % (w/w) sucrose (all sucrose solutions in
TE buffer,
see
Table
1
) into an empty tube.
(b) Underlay with 1.0 mL 60 % (w/w) sucrose.
(c) Overlay both fractions with 0.5 mL 50.5 % (w/w) sucrose.
(d) Overlay with 2.0 mL 48.5 % (w/w).
(e) Overlay with 1.0 mL 46.0 % (w/w).
(f) Overlay with 1.0 mL 43.7 % (w/w).
(g) Overlay with 1.0 mL 41.2 % (w/w).
Mark the fraction interfaces with a water-resistant pen.
One gradient is needed for the partially purifi ed leaf per-
oxisome fraction, while the second gradient is used as a
counterbalance. Store on ice until use.
12. Ice buckets containing ice.
13. Miracloth (Calbiochem Ltd., Nottingham, UK).
