Biology Reference
In-Depth Information
1. Transfer 20 mg of lyophilized plant material to a 2 mL
Eppendorf tube (
see
Note 4
).
2. Add 500
3.2.2 Procedure Based
on TCA-Acetone
Precipitation
L of 10 % (w/v) TCA, 0.07 % (w/v) DTT in 80 %
(v/v) acetone, and sonicate (three times × 10 s at 50 W, ampli-
tude 60) at 4 °C. Keep on ice for 1 min.
3. Mix vigorously by vortexing.
4. Fill the tube with 10 % (w/v) TCA, 0.07 % (w/v) DTT in
80 % (v/v) acetone, and vortex.
5. Allow to precipitate overnight at −20 °C.
6. Centrifuge at 16,000 ×
g
for 10 min at 4 °C and discard the
supernatant.
7. Wash the pellet (three times) with 80 % acetone.
8. Centrifuge at 16,000 ×
g
for 5 min at 4 °C and discard the
supernatant.
9. Air-dry the pellet to completely remove acetone.
10. Dissolve the pellet in 200
μ
μ
L of the solubilization solution for
4 h at 4 °C.
11. Quantify proteins using the Bradford method [
12
].
12. Store the extract at −20 °C for further analysis.
1. Transfer 20 mg of lyophilized plant material to a 2 mL
Eppendorf tube (
see
Note 4
).
2. Fill the tube with 10 % (w/v) TCA in 80 % (v/v) acetone and
sonicate (three times × 10 s at 50 W, amplitude 60, each) at
4 °C. Keep on ice for 1 min. Vortex vigorously.
3. Centrifuge at 16,000 ×
g
for 5 min and discard the
supernatant.
4. Fill the tube with 0.1 M ammonium acetate in 80 % (v/v)
methanol, and mix well by vortexing.
5. Centrifuge at 16,000 ×
g
for 5 min at 4 °C and discard the
supernatant.
6. Fill the tube with 80 % (v/v) acetone and mix well by
vortexing.
7. Centrifuge at 16,000 ×
g
for 5 min at 4 °C and remove the
supernatant.
8. Air-dry the pellet at room temperature to fully eliminate
acetone.
9. Fill the tube with the lysis-saturated phenol (1:1 ratio) solu-
tion, homogenize using a pestle, and keep on ice for 5 min.
10. Centrifuge at 16,000 ×
g
for 5 min at 4 °C and transfer the
upper phenolic phase into a new 2 mL tube.
3.2.3 Procedure Based
on TCA-Acetone
Precipitation and
Subsequent Extraction
with Phenol
