Biology Reference
In-Depth Information
portion (ca. 2-3 mL) of the (transparent) 38 % (v/v) Percoll
fraction. Carry out this step for
all
eight gradients (stored on
ice) before moving on to the next work-up step (
step 10
). In
this way residual thylakoids drain from the tube walls down-
wards and accumulate as a thin light green layer on top of the
remaining 38 % (v/v) Percoll fraction.
10. At this step, work up the SS34 tubes one by one until fi nal
transfer of the peroxisome fraction to the 100-mL collection
beaker. Carefully suck off the residual thylakoid layer, the 38 %
(v/v) Percoll fraction and the mixed 38 % (v/v) Percoll: 36 %
(w/w) sucrose fractions. Manually rotate the SS34 tube slowly
so that each gradient fraction is entirely removed. Only leave
about 2-3 mL 36 % (w/w) sucrose solution in each tube includ-
ing the leaf peroxisomes visible as whitish soft sediment at the
bottom of the gradient (Fig.
1
). Resuspend the peroxisome pel-
let gently with a disposable 3-mL plastic pipette of relatively
wide opening. Pool the eight peroxisome fractions in a (pre-
cooled) 100-mL beaker (ca. 16 mL in total). By this harvest
method, a post-centrifugation contamination of the leaf peroxi-
some fraction by the upper thylakoid fractions can be largely
avoided. A signifi cant chloroplast/thylakoid contamination is
indicated by a greenish color of the leaf peroxisome fraction.
11. To remove residual Percoll and fully adjust the sucrose concen-
tration to 36 % (w/w), dilute the leaf peroxisome fraction
approx. 1:4 very gently by mL-wise adding ca. 65 mL 36 %
(w/w) sucrose (in TE buffer) to a fi nal volume of about 80 mL.
12. Pour the diluted peroxisome fraction into four SS34 tubes and
centrifuge them at 39,000 ×
g
for 30 min.
13. Suck off the supernatant using a vacuum pump and carefully
collect the washed leaf peroxisome fraction (ca. 3-4 mL in
total) found at the bottom of the tubes by using disposable
3-mL plastic Pasteur pipettes with a wide opening to reduce
shear forces.
14. Homogenize the fraction carefully by approx. fi ve slow strokes
using a potter homogenizer. The homogenizer should have
moderate space between the pistil and the glass wall to avoid
application of damaging shear forces onto the leaf peroxisomes.
Transfer the fraction to a 10-mL measuring cylinder and adjust
the fi nal volume to 5 mL using 36 % (w/w) sucrose solution.
Add the three protease inhibitors at the given concentrations
as described above for the GB. This is the fi rst purifi ed leaf
peroxisome fraction (referred to as LP-P1 in Fig.
1
[
7
]), whose
purity and peroxisome intactness can be investigated by mea-
suring the activity of the leaf peroxisome marker enzyme HPR
(Table
2
) (
see
Note 13
).
15. Carefully lay the peroxisome fraction on top of the 41.2 %
(w/w) sucrose fraction of the preprepared sucrose density
