Biology Reference
In-Depth Information
6. 1 M DTT: Dissolve 1.54 g of DTT (
DL
-dithiothreitol) in
10 mL water. Dispense 50
L and store at −30 °C.
7. Dissolve 5.47 g of sorbitol in about 80 mL (
see
Note 6
), and
mix with 5 mL of 1 M HEPES-KOH, pH 7.5, 0.5 mL of 1 M
EDTA, 0.5 mL of 1 M MgCl
2
, 0.5 mL of 1 M MnCl
2
, and
5 mL of 1 M sodium ascorbate [
10
] or 50
μ
L of 1 M DTT (
see
Notes 7
and
8
). Make up to 100 mL with water. Stand on ice.
The prepared buffer should be used immediately.
μ
2.3 Plastid
Dilution Buffer
Dissolve 0.2 g of PVP with 5 mL of 1 M HEPES-KOH, pH 7.5,
0.5 mL of 1 M EDTA, and 0.5 mL of 1 M MgCl
2
, and then, make
up to 100 mL with water. Store at 4 °C.
1. 80 % Percoll: Mix 100 % Percoll with plastid isolation buffer in
the ratio of 4:1 by volume. Store at 4 °C.
2. 40 % Percoll: Mix 100 % Percoll with plastid isolation buffer and
distilled water in the ratio of 2:1:2 by volume. Store at 4 °C.
2.4
Percoll
2.5 Protein
Extraction Buffer
1. 1 M Tris-HCl, pH 8: Dissolve 60.6 g of Tris in 400 mL water
and adjust the pH with HCl (6N). Make up to 500 mL with
water. Store at 4 °C.
2. 50 % Glycerol: Mix 50 g of glycerol with water, and make up
to 100 mL with water.
3. 8 M urea: Dissolve 48.05 g of urea in 100 mL of water.
4. Dissolve 1.52 g of thiourea and 0.3 g of 3-[(cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHAPS) in about
2 mL water and mix with 1 mL of 1 M Tris-HCl, pH 8, 2 mL
of 50 % glycerol, 0.2 mL of Triton X-100, 0.2 mL of 1 M
DTT, and 3.75 mL of 8 M urea (
see
Note 9
). Make up to
10 mL with water. Stand on ice. The prepared buffer should be
used immediately.
2.6
MS Analysis
1. iTRAQ Reagents kit (AB SCIEX, CA, USA).
2. iCAT Cation Exchange Buffer Pack (Applied Biosystems, CA,
USA).
3. Cartridge holder (Applied Biosystems, CA, USA).
4. C-18 column (Sep-Pak C18 cartridges, Waters, Milford, MA,
USA).
5. 0.4 % formic acid (FA): Mix 80
L of formic acid with LC/
MS-grade water and make up to 20 mL in glass bottle.
6. 0.1 % FA: Mix 7.5 mL of 0.4 % FA and 22.5 mL of LC/
MS-grade water in glass bottle.
7. 75 % acetonitrile (ACN)/0.1 % FA: Mix 7.5 mL of 0.4 % FA
and 22.5 mL of ACN in glass bottle.
μ
