Biology Reference
In-Depth Information
5. Mortar and pestle.
6. Vortex.
7. Ultrasonic homogenizer.
8. Microcentrifuge.
9. Disposable microcentrifuge tubes: 1.5 and 2 mL.
10. PROTEAN II Cell and Protean Dodeca Cell (Bio-Rad,
Hercules, USA).
11. Protean IEF Cell system (Bio-Rad, Hercules, USA).
12. Micro-tubes mixer.
13. Gel shaker.
14. PowerPac 300 Power Supply (Bio-Rad, Hercules).
15. GS-800™ Calibrated Imaging Densitometer (Bio-Rad).
16. Quantity One
®
1-D Analysis software (Bio-Rad Hercules).
17. PD-Quest software v8.1 (Bio-Rad).
18. ProGest digestion station (Genomics Solution).
19. MALDI plates.
20. Automatic ProMs station (Genomic Solution).
21. Resin C18 microcolumn (ZipTip, Millipore).
22. 4800 Proteomics Analyzer (Applied Biosystems).
23. Autofl ex mass spectrometer (Bruker Daltonics).
24. MASCOT search engine (Matrix Science Ltd., London;
http://www.matrixscience.com
)
.
3
Methods
Plant material will be collected from different provenances or
genotypes. It is important to do a large sampling in order to have
the greatest diversity of populations or genotypes possible.
Undamaged, homogeneous mature acorns or pollen are collected
from at least ten different trees for each population. Once harvested,
plant material is put in airtight polyethylene bag, and then stored
at 4 ± 1 °C during no more than 12 h.
3.1 Plant Material
Collection and Storage
1. Immediately after arriving to the laboratory, and previously to
protein extraction, a pool of 20 acorns per tree are scarifi ed
with a knife by making transversal and longitudinal cuts, per-
mitting the rapid removal of the pericarp (
see
Note 6
).
2. After being peeled out, their embryos (including cotyledons)
are crushed in a blade mill (Moulinex AD56 42) until obtain a
fi ne powder (fl our).
3.1.1
Acorns
