Biology Reference
In-Depth Information
root etioplasts, the mechanism of mass transport in the amyloplasts
of reserve tissue, and the features of chromoplasts observed in fruit
and petals [
1
]. Proteomic technology is essential in these analyses,
and there are many cases in which the use of intact plastids for
proteomic analysis is indispensable.
Spinach, pea, and wheat leaves have been frequently used as
experimental materials in photosynthetic research on isolated
chloroplasts, because (1) these materials can be readily obtained,
(2) the leaves are soft, and (3) there are few compounds to inter-
fere with organelle isolation. With the rapid advances in genome
analysis, however, the need for organelle isolation from model
plants and crops, such as rice, maize, tomato,
Arabidopsis thaliana
,
and
Lotus japonicus
, has greatly increased [
2
-
9
]. Although varia-
tions in the size and shape of chloroplasts are observed in algae,
such variations are less pronounced in the case of higher plants,
with typical dimensions of 5-10
m in
thickness [
1
]. When isolating intact chloroplasts from higher
plants, it is necessary to consider the tissue organization and cell
components specifi c to the vegetable species rather than the size
and shape of the chloroplasts.
Here, a typical procedure for the isolation of intact plastids,
mainly focusing on chloroplasts, as well as a method of trouble-
shooting are described. We also outline a well-established compre-
hensive shotgun proteomic analysis technique using the iTRAQ
labeling method to characterize the characteristics of intact
plastids.
μ
m in diameter and 3-4
μ
2
Materials
Fresh and soft tissue should be selected as the material. The mixing
of starch and inorganic substances should be avoided at the step of
plastid isolation (
see
Notes 1
-
3
).
2.1 Sample
Materials
1. 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES)-KOH, pH 7.5: Dissolve 23.8 g of HEPES in about
80 mL of water and adjust the pH with KOH (10N). Make up
to 100 mL with water. Store at 4 °C (
see
Note 4
).
2. 1 M ethylenediaminetetraacetic acid (EDTA): Dissolve 37.2 g
of EDTA 2Na·2H
2
O in about 80 mL of water and adjust pH
to 8 with KOH (10N). Make up to 100 mL with water. Store
at 4 °C (
see
Note 5
).
3. 1 M MgCl
2
: Dissolve 20.3 g of MgCl
2
·6H
2
O and make up to
100 mL with water. Store at 4 °C.
4. 1 M MnCl
2
: Dissolve 12.6 g of MnCl
2
and make up to 100 mL
with water. Store at 4 °C.
5. 1 M sodium ascorbate: Dissolve 19.8 g of sodium
L
-ascorbate
and make up to 100 mL with water. Store at 4 °C.
2.2 Plastid
Isolation Buffer
