Suspension-Cultured Plant Cells as a Tool to Analyze the Extracellular Proteome - Plant Proteomics: Methods and Protocols - page 398

2. Once the fruits are disinfected, and working in the laminar
air-fl ow chamber, remove calcium hypochlorite. Add sterile
distilled water and wash three times for 1 min each time.
3. Divide the immature fruit berries in four portions with the
help of a scalpel, working on sterile Petri dish.
4. Transfer the explant into calli-induction medium, and place
Petri dishes at 25 °C in darkness ( see Note 13 ). Subculture
cultures every 3 weeks until they reach approximately 3 cm in
size.
5. After callus formation, the different callus tissues are separated
from each explant and transferred to 250 mL sterile Erlenmeyer
fl asks containing 100 mL of solid growth medium. Both
explants and calli are maintained at 25 °C in darkness, and sub-
cultured for 2-3 weeks.
6. Initiate grapevine SCC by transferring 20 g of fresh-weight
friable calli into 250 mL Erlenmeyer fl asks containing 100 mL
of liquid growth medium. Maintain SCC in a rotary shaker
(110 rpm) at 25 °C in darkness. Subculture SCC every 14 days
by diluting with one volume of liquid growth medium and
then distributing into two fl asks.
7. Separate cells from the extracellular medium by fi ltration using
a gentle vacuum. This extracellular medium is used for protein
precipitation.

1. SCC is fi ltrated through a glass vacuum fi lter and the extracel-
lular medium is kept for further analysis.
2. The cell-free medium is frozen at −20 °C overnight. Then, the
extracellular medium is thawed and centrifuged at a medium
speed (8,000 × g (Sorvall RC-5B plus, SS-34 rotor)) in order
to remove polysaccharides.
3. Add ethyl acetate to the supernatant (1:4, v/v) to remove stil-
benes ( see Note 14 ). Stir for 30 min and centrifuge at 6,700 × g.

3.2 Protein Sample
Preparation

4. After removing the organic phase, the aqueous phase is supple-
mented with 2 % (w/v) PVPP and incubated for 60 min with
shaking. The slurry is centrifuged at 8,000 × g for 15 min to
remove the insoluble PVPP and the supernatant is kept for
protein extraction ( see Note 15 ).
5. The proteins are recovered by precipitation adding TCA to a
fi nal concentration of 6 % (w/v) and pelleted at 13,400 × g
during 10 min ( see Notes 16 and 17 ).
6. The pellets are washed in ice-cold methanol which means that
the pellet is resuspended and then centrifuged at 13,400 × g .
Repeat the sequence twice.
7. The pellet is then washed in ice-cold acetone three times as
described above.