Biology Reference
In-Depth Information
Fig. 3
Venn diagram of B05.10 versus T4
B. cinerea
strains of proteins identifi ed by
using gel-free approach from (
a
) mycelium extracts and (
b
) secreted proteins
2
Materials
This protocol has been carried out with different
B. cinerea
strains:
B05.10 and T4 (provided by Dr. Julia Schumacher, Prof. Dr. Paul
Tudzynski of the Institute of Biology and Biotechnology of Plants,
Westfälische Wilhelms-Universität, Münster, Germany), together
with CECT2100, CECT2850, CECT2996, and CECT20518
(provided by the Spanish Type Culture Collection).
2.1 Fungal Strains
Analytical grade reagents are used, unless other grades are specifi ed.
The prepared solutions are kept at 4 °C or −20 °C if indicated.
Reagents and solutions must be discarded once used, according to
current regulations. It is mandatory to use a fume cupboard when
working with volatile or dangerous compounds. Personal protection
elements (i.e., lab coats, gloves, glasses) must be used (
see
Note 1
).
2.2 Reagents,
Solutions, and Buffers
1. 10 % (w/v) TCA in 80 % (v/v) acetone.
2. 0.1 M ammonium acetate in 100 or 80 % methanol.
3. 80 % (v/v) acetone.
4. Phenol solution equilibrated with 10 mM Tris-HCl, pH 8
(P4557, Sigma).
5. SDS buffer: 0.1 M Tris-HCl, pH 8, 30 % (w/v) sucrose, 2 %
(w/v) SDS, 5 % (v/v)
-mercaptoethanol.
6. Solubilization solution: 9 M urea, 2 M thiourea, 4 % (w/v)
CHAPS, 0.5 % (v/v) Triton X-100, 20 mM DTT.
7. Bradford solution (B6916, Sigma).
8. Extraction buffer: 50 mM Tris-HCl, pH 8, 8 M urea, 1 %
(w/v) SDS, 1 mM EDTA, 100 mM DTT.
9. TE buffer for secreted proteins: 10 mM Tris-HCl, pH 8, 1 mM
EDTA, 2 % (v/v)
β
L/mL
buffer of protease inhibitor cocktail for fungi (P8215, Sigma).
β
-mercaptoethanol, 1 mM PMSF, 10
μ
