Biology Reference
In-Depth Information
-Aminocaproic acid (in H
2
O, freshly prepared)
are recommended. Instead of this self-mixed protease inhibitor
set, alternatives can be used such as the Complete Protease
Inhibitor Cocktail Tablets (Roche).
5. Measure exactly the required Percoll volume and the fi nal solu-
tion volume because the fi nal Percoll concentration of 15 %
(v/v) crucially determines peroxisome yield and purity.
6. If preparing the Percoll solutions directly in TE buffer, pH 7.5,
the pH needs to be readjusted back to 7.5 because the pH of
Percoll (ca. 9) increases the pH of the TE buffer.
7. The sucrose concentration (in % w/w) needs to be adjusted to
highest accuracy at 20 °C using a refractometer.
8. The density of the 38 % (v/v) Percoll solution and the 36 %
(w/w) sucrose in TE buffer solution differ only marginally
from each other but are prerequisite for proper gradient prepa-
ration and leaf peroxisome isolation. Prior to gradient assem-
bly it is recommended to check in an Eppendorf tube whether
the density of the 36 % (w/w) sucrose solution is indeed
slightly higher than that of the 38 % (v/v) Percoll solution by
either underlying or overlaying 0.5 mL of one solution with
0.5 mL of the other. If positioning of the two solutions is dif-
fi cult to see, one solution aliquot can be stained blue with a
grain of Coomassie Brilliant Blue (e.g., R250).
9. Store the gradients on ice for at least 30 min before use.
10. If required, the total gradient volume can be reduced by reduc-
ing the volume of the lower fractions, i.e., 0.8 mL 60.0 %
(w/w) and 0.8 mL 55.2 % (w/w) sucrose.
11. The plants should best be kept in the dark for about 12 h
before starting the peroxisome isolation. This extended dark
incubation lowers the physical adherence between leaf peroxi-
somes, chloroplasts, and mitochondria and signifi cantly reduces
the contamination of leaf peroxisomes by chloroplasts and
mitochondria. Also the post-harvest leaf incubation on ice
appears to reduce inter-organelle adherence.
12. Mix the crude extract by gently shaking the Erlenmeyer fl ask,
determine the total volume of the crude extract (approx. 140 mL),
take small aliquots for organelle enrichment analyses (Table 2)
(e.g., 3 × 1 mL), and freeze them at −20 °C.
and 200 mM
ɛ
13. In Arabidopsis, two additional HPR homologues have recently
been shown to account for minor NAD(P)H-dependent HPR
activities in the cytosol and chloroplasts, respectively [
17
,
18
],
but are not considered to signifi cantly diminish the traditional,
easy and reliable use of peroxisomal HPR as a marker enzyme
for leaf peroxisomes.
14. Start measuring the 1-min centrifugation when the maximum
speed has been reached.
