Biology Reference
In-Depth Information
3. Centrifuge at 16,000 ×
g
for 5 min (4 °C).
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 discard the
supernatant.
6. Fill the tube with 0.1 M ammonium acetate in 80 % (v/v)
methanol. Mix well using fi rstly a micropestle, and secondly by
vortexing.
7. Follow the steps in Subheading
3.2.1
(starting from
step 5
).
3.3 Protein
Samples for LC
The fungal protein extracts obtained by the TCA/acetone-phenol/
methanol method are re-solubilized in a solubilization solution that
contains thiourea, CHAPS, and Triton X-100. These compounds
are important for protein solubilization, but they interfere with
many downstream analysis methods (as is the case of LC separation).
For this reason, it is crucial to remove them. Several methods to
solve this problem are described below:
1. Protein solubilization with a solution of 6 M urea and 50 mM
(NH
4
)
2
CO
3
instead of the solubilization solution (
see
Note 8
).
2. Use of cleaning kits (e.g., 2-D Clean-up kit; GE Healthcare),
and protein re-solubilization in detergent-free solutions (i.e.,
6 M urea, 50 mM (NH
4
)
2
CO
3
) (
see
Note 8
).
3. Use of electrophoresis using 5 % acrylamide gels. Proteins
(about 50-100
g) are loaded onto the gel, run at 100 V,
stained using the Coomassie method, and fi nally the protein
band is cut out (
see
Note 9
).
μ
3.4 Protein
Separation by 1-DE
Proteins can be separated by SDS-PAGE with the Laemmli electro-
phoresis system [
68
], for example using the Criterion System (Bio-
Rad) with precast Criterion Stain-Free precast Gels, Tris-HCl, and
4-20 % linear gradient (Bio-Rad). The 1-DE is visualized using the
Image Lab System (Bio-Rad), and stained by Coomassie Blue
Brilliant (CBB) method [
69
] (
see
Note 10
). After protein staining,
bands can be analyzed using the Quantity One software (Bio-Rad).
3.5 Protein
Separation by 2-DE
Focusing conditions will vary with sample composition, complexity,
and strip pH range. In our conditions, the 11 cm IPG strips, pH 5-8
(Bio-Rad), are rehydrated with 50
g of protein extract in rehydra-
tion solution according to the manufacturer's instructions, and
applying 50 V for 16 h (active rehydration) at 20 °C. Before this
focusing, a wet wick is placed under each end of the strip (cathode).
The conditions for IEF have been adapted to our system from [
38
]:
150 V for 1 h, 1 h at 200 V, 1 h at 500 V, 1,000 V h at 1,000 V,
followed by 2.5-h gradient from 1,000 to 8,000 V, and fi nally
focused for 20,000 V·h at 8,000 V, with a cell temperature of 20 °C
(
see
Note 11
). After IEF, IPG strips are stored at −20 °C.
μ
