Biology Reference
In-Depth Information
19. Air-dry the pellet at room temperature.
20. Dissolve the proteins with a solubilization solution by shaking
for 2 h in a microtube mixer at 4 °C (
see
Note 6
).
21. Quantify proteins using the Bradford method [
67
].
22. Store the protein extracts at −20 °C for further analysis.
Lyophilized media are re-solubilized in 5 mL of TE buffer, and
proteins are precipitated using the following protocol:
3.2.2 Secreted Proteins
1. Transfer the re-solubilized medium into a 50-mL tube and add
2/1 (v/v) (10 mL) of 20 % (w/v) TCA/acetone. Mix well by
vortexing and allow protein precipitation overnight at 4 °C.
2. Centrifuge at 16,000 ×
g
for 10 min (4 °C) and remove the
supernatant by decanting (
see
Note 7
).
3. Add 4/1 (v/v) (20 mL) of 0.1 M ammonium acetate in 80 %
(v/v) methanol. Mix well by vortexing.
4. Centrifuge at 16,000 ×
g
for 10 min (4 °C) and discard the
supernatant.
5. Add 4/1 (v/v) (20 mL) of 80 % (v/v) acetone. Mix well by
vortexing.
6. Centrifuge at 16,000 ×
g
for 10 min (4 °C) and discard the
supernatant.
7. Air-dry the pellet at room temperature to remove residual acetone.
8. Add 1.5 mL of 1/1 (v/v) phenol (pH 8, Sigma)/SDS buffer.
Mix well by vortexing and transfer the results into a new 1.5-
mL Eppendorf tube. Incubate for 5 min on ice.
9. Centrifuge at 16,000 ×
g
for 10 min. Transfer the upper phe-
nol phase into a new 2-mL tube.
10. Fill the tube with 0.1 M ammonium acetate in 100 % (v/v) metha-
nol, mix well, and allow the precipitation overnight at −20 °C.
11. Follow the steps in Subheading
3.2.1
(starting from
step 15
).
Conidia can be harvested from H
2
O with 0.01 % Tween-80
scraping on the surface of an agar plate. The conidial suspension
is fi ltered through a cell strainer, concentrated in 1.5-mL tubes,
centrifuged at 16,000 ×
g
for 5 min (4 °C), lyophilized, and stored
at −80 °C for further analyses. For protein extraction, a buffer-
based extraction is used and the proteins are precipitated using
the TCA/acetone-phenol/methanol method [
36
,
66
] with some
modifi cations [
12
,
20
]:
1. Add 300
3.2.3 Conidia
L of buffer extraction to conidia. Mix well fi rstly
using a micropestle, and secondly by vortexing.
2. Sonicate 3 × 10 s (50 W, amplitude 60), breaking on ice at 1 min.
Mix well fi rstly using a micropestle, and secondly by vortexing.
μ
