Biology Reference
In-Depth Information
7. Preparative centrifuge with rotors capable of processing
2 × 500 mL samples at 800 ×
g
and 4 × 50 mL samples at
10,000 ×
g
.
8. Ultracentrifuge with fi xed angle rotor capable of processing
4 × 12 mL samples at 100,000 ×
g
.
9. 5 kDa Ultrafree centrifugal fi lter devices (EMD Millipore,
MA, USA) for protein concentration.
10. Tandem mass spectrometer (MS/MS) coupled with online
liquid chromatography (LC) (nanofl ow or capillary fl ow rates)
capable of data dependent acquisitions.
11. Tandem mass spectrometer (MS/MS) coupled with online
liquid chromatography (LC) capable of undertaking selected
reaction monitoring techniques (e.g., triple quadrupole mass
spectrometer).
12. Collection of plant organelle marker antibodies (Agrisera AB,
Vännäs, Sweden).
2.2 Purifi cation of
the Cytosolic Fraction
from Plant Seedlings
1. Plant material: 14-day-old
Arabidopsis
grown in 2.2 g/L
Murashige and Skoog (MS) medium, 1 % (w/v) sucrose, and
0.7 % (w/v) agar at 100
μ
mol photons/m
2
s for a 12 h photo-
period at 24 °C.
2. Digestion Medium: 1.5 % (w/v) cellulase (Yakult
Pharmaceutical, Tokyo, Japan), 0.4 % (w/v) macerozyme
(Yakult Pharmaceutical, Tokyo, Japan), 0.5 M sucrose, 20 mM
KCl, 10 mM CaCl
2
, and 20 mM MES-KOH, pH 5.7. Dissolve
enzymes in the solution until it becomes clear light brown and
fi lter through 0.45
m syringe fi lter device (
see
Note 3
).
3. Wash Buffer: 0.5 M sucrose, 20 mM KCl, 10 mM CaCl
2
, and
20 mM MES-KOH, pH 5.7.
4. Flotation Medium I (FMI): 0.5 M Sucrose, 1 mM MgCl
2
, and
5 mM HEPES, pH 7.0 (
see
ref.
28
).
5. Flotation Medium II (FMII): 0.4 M sucrose, 0.1 M sorbitol,
1 mM MgCl
2
, and 5 mM HEPES, pH 7.0 [
28
].
6. Flotation medium III (FMIII): 0.5 M sorbitol, 1 mM MgCl
2
,
and 5 mM HEPES, pH 7.0 [
28
].
7. Chloroplast Buffer: 50 mM HEPES-KOH, pH 8.0, 5 mM
EDTA, 5 mM EGTA, 330 mM sorbitol, 5 mM cysteine, and
5 mM ascorbic acid.
8. Percoll Solution: 95 % (w/v) Percoll™, 3 % (w/v) PEG 6000,
1 % (w/v) Ficoll™ [
12
] (
see
Note 4
).
9. Gradient Mixture: 25 mM HEPES-NaOH (pH 8.0), 10 mM
EDTA, 5 % (w/v) sorbitol [
12
].
10. Glass-Tefl on Potter-Elvehjem Tissue Grinders (10-20 mL
capacity), cooled on ice.
μ
