Biology Reference
In-Depth Information
3.4 PEG
Fractionation for
Removal of RuBisCO
This method is depicted as a fl ow chart in Fig.
1
.
1. Incubate fi nely powdered tissue sample with Mg/NP-40
extraction buffer (5 mL/g tissue).
2. Mix for 30 min on ice and centrifuge at 12,000 ×
g
for 15 min.
3. Transfer the supernatant into a new 50 mL Nalgene centrifuge
tube.
4. Add PEG to a fi nal concentration of 15 % (w/v) using a 50 %
(w/v) PEG stock solution and incubate on ice for 30 min or
more (
see
Note 8
).
5. Centrifuge at 12,000 ×
g
for 15 min at 4 °C.
6. Save the PEG pellet. Transfer supernatant to a new tube and
add 4 volumes of 100 % acetone.
7. Place at −20 °C for 2-3 h to precipitate proteins and then
centrifuge at 3,000 ×
g
for 15 min.
8. Resuspend the PEG pellets in 10 mL of Mg/NP-40 extraction
buffer and vortex at RT.
9. Add an equal volume of water-saturated phenol, vortex, and
centrifuge at 3,000 ×
g
for 15 min.
10. Collect the phenolic phase and precipitate proteins by adding
4 volumes of methanol containing 0.1 M ammonium acetate
at −20 °C for 3 h followed by centrifugation at 3,000 ×
g
for
10 min.
11. Decant solution carefully and wash protein pellet three times
with methanol containing 0.1 M ammonium acetate. Store in
80 % acetone at −20 °C until use.
12. Proceed for 2-DE analysis (
see
Note 11
).
3.5 In Planta
Secretome from Rice
Leaf Infected with
M. oryzae or X. oryzae
This method is depicted as a fl ow chart in Fig.
2
.
1. Cut rice leaves (length of average 5 cm)-infected with/without
M. oryzae
or
X. oryzae
(approx. 50 g) with scissors (
see
Note 9
).
2. Place cut leaves in a 250 mL centrifuge bottle containing
150 mL of CA buffer (
see
Note 10
) and hold on a constant
horizontal shaker at 100 strokes/min for 1 h on ice to extract
secreted proteins.
3. Filter the extraction buffer with No.2 fi lter paper to remove
leaf debris.
4. Centrifuge at 2,500 ×
g
for 15 min at 4 °C and transfer the buffer
to a new centrifuge tube.
5. Add 50 mL of water-saturated phenol to the extracted super-
natant solution, mix, and centrifuge at 5,000 ×
g
for 15 min at
RT, and transfer phenolic phases to new tube. Save superna-
tant solution and repeat this step once more to collect enough
protein combine phenol fractions.
