Biology Reference
In-Depth Information
11. In the case of monocotyledons, remove the midrib and veins of
the leaf beforehand [ 16 ].
12. Perform the steps from extraction to centrifugation quickly for
best results. If the sample turns brown, add 5-20 mM of a
reducing agent such as DTT or ascorbic acid to the extraction
solution.
13. It is desirable to perform all of the steps in low-light condition
to reduce the risk of plastid destruction.
14. For plants such as rice, due to the formation of calcium car-
bonate crystals, it is necessary to perform tissue disruption
gently. Chopping with a razor blade may increase the effi -
ciency of extraction of intact chloroplasts. The use of a
chilled blender can increase the yield of chloroplasts, but
with lower purity.
15. Increasing the centrifugal force (maximum 8,000 × g ) results in
a higher yield of chloroplasts but also increases contamination
from broken organelles.
16. The pelleted chloroplasts are easily damaged. Thus, the buffer
solution should be applied to the pellet using a pipette tip with
the end cut, allowing the suspension to be formed by the pres-
sure of the applied solution.
17. Nycodenz (Axis-Shield PoC AS, Oslo, Norway) can also be
used instead of Percoll [ 17 ]. Nycodenz has a high separation
capacity, low cellular toxicity, and no enzyme activity
inhibition.
18. Intact chloroplasts can be easily observed under a light micro-
scope. An intact plastid has a globular shape with glittering
surroundings, whereas a broken chloroplast has a distorted
shape with a dark green interior.
19. Chloroplasts easily break and their intactness cannot be
maintained for a prolonged period even on ice. They are
also destroyed by freezing and thawing. It is recommended
that isolated chloroplasts be kept in a sucrose solution
(0.5-1 M).
20. A large amount of pigment affects the effi ciency of protein
identifi cation by LC-MS/MS. Therefore, it should be removed
completely by TCA/acetone precipitation beforehand.
21. Urea is effective in dissolving protein aggregation, but its fi nal
concentration should be kept to less than 1 M.
22. The protein samples should be adjusted with dissolution buffer
to an equal concentration.
23. If the sample contains no urea, incubate at 60 °C.
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