Biology Reference
In-Depth Information
Table 1
Preparation of different sucrose density gradient fractions from stock solutions
Final sucrose
conc. (% (w/w))
Final
volume (mL)
Volume of 60 % (w/w)
sucrose sol. (mL)
Volume of 36 % (w/w)
sucrose sol. (mL)
41.2
50
10
40
43.7
50
15
35
46.0
50
20
30
48.5
50
25
25
50.5
50
35
15
55.2 50 40 10
From two sucrose stock solutions of 60 % (w/w) and 36 % (w/w) different working solutions required for the sucrose
density gradient for Arabidopsis leaf peroxisome isolation are prepared in 50-mL Falcon tubes and stored in the refrig-
erator. Note that mixing of the two stock solutions in the given proportions only roughly yields the desired sucrose
concentration of the working solutions. The precise sucrose concentration needs to be adjusted by addition of a small
volume of appropriate stock solution using a refractometer
5.0 mL leaf peroxisome fraction LP-P1
40 min to 2 h at 80,000x
g
1.0 mL 41.2% (w/w) sucrose
1.0 mL 43.7% (w/w) sucrose
1.0 mL 46.0% (w/w) sucrose
2.0 mL 48.5% (w/w) sucrose
Leaf
peroxisomes
0.5 mL 50.5% (w/w) sucrose
1.0 mL 55.2% (w/w) sucrose
1.0 mL 60.0% (w/w) sucrose
Fig.
2
Leaf peroxisome enrichment from Arabidopsis by sucrose density gradient
centrifugation (second density gradient). The gradient consists of seven different
sucrose fractions (41.2 %
top
to 60.0 % (w/w)
bottom
). After centrifugation
(40 min to 2 h at 80,000 ×
g
) the leaf peroxisomes are located at the bottom of the
centrifugation tube at the interface between 50.5 and 55.2 % (w/w) sucrose. The
fi gure has been adapted and reprinted from Reumann et al. [
7
] Suppl. Figure 1
14. Sorvall centrifuge RC-5C with SS34 rotor.
15. Ultracentrifuge with swinging bucket rotor (e.g., SW41 Ti).
16. Ultrapure water.
17. 20-mL glass pipettes and Pasteur pipettes (disposable).
