Biology Reference
In-Depth Information
presents particular challenges (5-10 mg mitochondrial
protein from 50 to 100 g FW, 100
μ
g/g FW) [
2
,
12
,
35
,
46
].
●
Certain tissues, such as stems, seeds, succulents, embryos,
and fruits, are often more diffi cult to isolate mitochondria
from although it is possible, but specialized techniques are
required and these are beyond the scope of this chapter.
The power of proteomics is largely dictated by the
availability of protein sequences from the species being
studied, so therefore species with high-quality publicly
available genome sequences such as Arabidopsis and rice
are preferred for proteomics investigations due to the ease
of analysis and interpretation of mass spectrometry data.
However species with un-sequenced genomes can be
matched against EST databases or matched across species
to plant databases (i.e., NCBInr—Viridiplantae), although
the limitations of these approaches should be fully appre-
ciated before proceeding [
47
].
2. Homogenization solution: 0.4 M sucrose (
see
Note 1
), 5 mM
EGTA (
see
Note 2
), 50 mM sodium pyrophosphate-KOH,
pH 7.5 (
see
Note 3
), 0.5 % (w/v) BSA (
see
Note 4
), 10 mM
ascorbate (
see
Note 5
), and 1 % (w/v) PVP-40 (
see
Note 6
).
Prepare this media 1 day before the isolation and store it over-
night at 4 °C (
see
Note 7
).
3. Wash solution (WS): 10 mM TES-KOH, pH 7.2, 0.3 M
sucrose, and 0.1 % (w/v) BSA.
4. Buchner funnel.
5. Sterile distilled water.
6. Perspex vessel (45 × 60 × 220 mm).
7. Polytron
®
homogenizer equipped with a 200 mm long × 20 mm
diameter dispersing head (
see
Note 8
).
8. Miracloth (Merck Millipore).
9. Muslin.
10. 50 mL centrifuge tubes.
11. Centrifuge with fi xed-angle rotor (Beckman Coulter Avanti
J-26XP with JA-25.50 rotor or an equivalent).
12. Soft-haired artist's paintbrush.
13. 28 % (v/v) Percoll
®
, 0-4.4 % (w/v) PVP-40 gradients (
see
Subheading
3.2.3
).
14. 3 mL disposable pipette.
2.3 Determining
Mitochondria Quality
1. Mitochondria isolated according to Subheading
3.2
.
2. Clark-type electrode, such as those manufactured by Hansatech
(Kings Lynn, UK) or Rank Brothers (Bottisham, UK).
