Plant Mitochondrial Proteomics - Plant Proteomics: Methods and Protocols - page 489

3. The supernatant is gently transferred into another set of
centrifuge tubes without disturbing the pellet containing
starch, nuclei, and cell debris. The supernatant is centrifuged
at 21,000 × g for 20 min at 4 °C.
4. Discard supernatant. The pellet containing crude mitochon-
dria is resuspended in ~4.0 mL of wash solution with the aid
of a clean soft-bristle artist's paintbrush. The resuspended
organelles are transferred to 50 mL centrifuge tubes, the vol-
ume adjusted to 40 mL with more WS, and centrifuged at
1,500 × g for 5 min at 4 °C.
5. The supernatants are transferred into another set of tubes and
centrifuged at 21,000 × g for 20 min at 4 °C. The resulting
pellet containing crude organelles can be uniformly resus-
pended in a small volume (~2.0 mL) of WS with the aid of a
clean soft-bristle paintbrush.

This crude mitochondrial fraction is still heavily contaminated and
remains unsuitable for proteomics. The mitochondria are further
enriched using step or continuous gradient centrifugation (Fig. 1 ).
We employ Percoll ® step gradients for non-photosynthetic tissues
and Percoll ® continuous gradients with 0-4.4 % (v/v) PVP-40 for
photosynthetic tissues. Once the mitochondrial fraction has been
collected from the gradient, two wash steps are required to dilute
the concentrations of Percoll ® and PVP by at least tenfold. This is
followed by a second self-forming 28 % Percoll ® gradient and
washes to further purify mitochondria.

3.2.3 Density Gradient
Centrifugation

1. The washed mitochondria are layered over 35 mL of the cho-
sen gradient solution in a 50 mL centrifuge tube ( see Note 18 )
and then centrifuged at ~40,000 × g for 45 min at 4 °C with
the brake off during deceleration ( see Note 19 ).
2. After centrifugation, the less dense layers of the gradient con-
taining broken thylakoid membranes are discarded by aspira-
tion. The mitochondria should form a yellow-brown band
toward the bottom of the tube. Collect this band with a Pasteur
pipette, taking care not to collect the very dense matter pooled
at the bottom of the tube. The collected mitochondria are
diluted with at least 4 volumes of WS without BSA, and centri-
fuged at 21,000 × g for 15 min at 4 °C in 50 mL tubes.

3. The resulting loose pellet is resuspended in WS without BSA
and centrifuged again at 21,000 × g for 15 min. The mito-
chondrial pellet is resuspended in a small volume of WS
(~1 mL) without BSA.
4. This pellet is then layered on top of a 28 % (v/v) Percoll ® solu-
tion for a second gradient step in 50 mL centrifuge tube and
then centrifuged at ~40,000 × g for 30 min at 4 °C with the
brake off during deceleration ( see Note 19 ).