Biology Reference
In-Depth Information
10. Electrophoresis buffer: 50 mM Tris-HCl, pH 8, 192 mM gly-
cine, 1 % (w/v) SDS.
11. Stain-Free Precast Gels (Criterion System, BioRad): 4-20 % Tris-
HCl multi-wells for 1-DE and 8-16 % Tris-HCl IPG+1 for 2-DE.
12. IPG strips, 11 cm, pH 5-8 (BioRad).
13. IPG strip rehydration solution: 7 M urea, 2 M thiourea, 4 %
(w/v) CHAPS, 2 % (v/v) ampholytes (BioRad), 20 mM DTT.
14. Equilibration buffer: 1.5 M Tris-HCl, pH 8.8, 6 M urea, 20 %
(v/v) glycerol, 2 % (w/v) SDS.
15. Distilled water.
16. Liquid nitrogen.
2.3 Equipment
1. Freeze-dryer.
2. Mortar and pestle.
3. Cell strainer, 100
μ
m nylon.
4. Vortexer.
5. Micropestles.
6. Ultrasonic homogenizer.
7. Microcentrifuge and centrifuge.
8. Disposable microcentrifuge tubes: 1.5 and 2.0 mL.
9. Centrifuge tubes: 50 mL.
10. Microtube mixer.
11. Criterion™ Cell (Biorad).
12. Criterion Stain Free Imager and Image Lab™ software (Biorad).
13. Shaker.
14. GS-800™ Calibrated Densitometer and Quantity One
®
1-D
Analysis software (BioRad).
3
Methods
The methods described below have been optimized to mycelium,
secreted proteins in liquid media, and conidia from
Botrytis cine-
rea
, although these procedures can be applied to proteomic analy-
sis of fi lamentous fungi in general.
3.1 Sample
Collection
For in vitro cultures, conidia are produced using rich-media plates
at 22 °C under constant black light (UV) during 3-4 weeks.
Mycelium and secreted proteins can be obtained from liquid cultures
inoculated with conidia or non-sporulating mycelia (
see
Note 2
).
Mycelia and media can be separated by centrifugation and fi ltration,
frozen in liquid nitrogen, or lyophilized. At least three biological
replicates should be collected [
65
].
