Biology Reference
In-Depth Information
3.2 Protein
Extraction by TCA/
Acetone-Phenol/
Methanol
Protein extraction is carried out by using the TCA/acetone-phenol/
methanol method [
36
,
66
] with some modifi cations [
12
,
45
], and
adapted to the initial material (conidia, mycelium, or secreted
proteins).
The lyophilized mycelium is ground to a fi ne powder in liquid
nitrogen using a cooled mortar and pestle, and then it is stored at
−80 °C for later analysis (
see
Note 3
). The following protocol is
used for protein extraction:
3.2.1 Mycelium
1. Transfer 50-100 mg of mycelial powder into a 2-mL tube.
2. Add 1 mL of 10 % (w/v) TCA/acetone and mix well fi rstly
using a micropestle, and secondly by vortexing.
3. Sonicate 3 × 10 s (50 W, amplitude 60) at 4 °C, breaking on
ice at 1 min.
4. Fill the tube with 10 % (w/v) TCA/acetone. Mix well by
vortexing.
5. Centrifuge at 16,000 ×
g
for 5 min (4 °C) and remove the
supernatant by decanting (
see
Note 4
).
6. Fill the tube with 0.1 M ammonium acetate in 80 % (v/v)
methanol. Mix well by vortexing.
7. Centrifuge at 16,000 ×
g
for 5 min (4 °C) and discard the
supernatant.
8. Fill the tube with 80 % (v/v) acetone. Mix well by vortexing.
9. Centrifuge at 16,000 ×
g
for 5 min (4 °C) and discard the
supernatant.
10. Air-dry the pellet at room temperature to remove residual
acetone.
11. Add 1.2 mL of 1:1 phenol (pH 8, SIGMA)/SDS buffer. Mix
well by vortexing and using a pipette. Incubate for 5 min on ice.
12. Centrifuge at 16,000 ×
g
for 5 min. Transfer the upper phenol
phase into a new 1.5-mL tube (
see
Note 5
).
13. Fill the tube with 0.1 M ammonium acetate in 100 % (v/v)
methanol, mix well, and incubate the precipitation overnight
at −20 °C.
14. Centrifuge at 16,000 ×
g
for 5 min (4 °C) and discard the
supernatant (a white pellet should be visible).
15. Wash the pellet with 100 % methanol, and mix by vortexing.
16. Centrifuge at 16,000 ×
g
for 5 min (4 °C), and discard the
supernatant.
17. Wash the pellet with 80 % (v/v) acetone, and mix by
vortexing.
18. Centrifuge at 16,000 ×
g
for 5 min (4 °C), and discard the
supernatant.
