Biology Reference
In-Depth Information
3. The fi nal powder is weighted and stored in a desiccator at
4 ± 1 °C until protein extraction.
4. For SDS-PAGE protein extraction, fl our of ten independent
samples per population are used; whereas for 2-DE protein
extraction fl our representing all studied accessions of each
population are crushed together and its proteins are extracted.
1. Immediately after arriving to the laboratory, pollen grains are
isolated from freshly open fl owers by shaking the anthers on a
glass slide (
see
Note 6
).
2. Flower debris is removed using a microsieve, and pollen is
examined under a light microscope.
3. Pollen is either used immediately, or stored at −70 °C after
freezing in liquid nitrogen (
see
Note 7
).
3.1.2
Pollen
3.2 Protein
Extraction from
Acorns by the TCA/
Acetone Method
To extract protein from acorns, we propose to use the procedure
suggested by Damerval et al. [
14
]. It is particularly suitable for the
extraction of proteins from seeds, but also for leaves as reported by
Jorge et al. [
31
] and Maldonado et al. [
13
]. The method described
here has been optimized to leaves and seeds from
Q. ilex
subsp.
ballota
[
15
], although these procedures can be applied to plant
proteomic analysis in general.
1. The powder (fl our) (100 mg) is transferred into a 2 mL tube
with 1 mL of a solution of 10 % (w/v) TCA/acetone with
0.07 % (w/v) DTT. Mix well using a micropestle and then by
vortexing (
see
Notes 6
and
8
).
2. Sonicate 3× 10 s (50 W, amplitude 60) at 4 °C.
3. The proteins are left to precipitate overnight and then centri-
fuged at 15,000 ×
g
at 4 °C for 15 min. Discard the
supernatant.
4. The pellet is washed twice with 1 mL of a solution of acetone
with 0.07 % (w/v) DTT, and then centrifuged at 15,000 ×
g
at
4 °C for 15 min. Discard the supernatant.
5. The pellet obtained is dried in the air in order to remove resid-
ual acetone (
see
Note 9
).
6. Proteins are dissolved in a solubilization solution for 2 h, by
shaking in a microtube mixer at 4 °C (
see
Note 10
).
7. Proteins are quantifi ed using the Bradford method [
32
].
Prepare the calibration curve using several dilutions of bovine
serum albumin protein, containing concentration of 0, 1, 3, 5,
10, 15, and 20
L of BSA (1 mg/mL) into 1.5 mL tubes, and
make all up to 500
μ
L of
Bradford reagent to each tube and mix well by vortexing gen-
tly for thorough mixing. Use 800
μ
L with distilled water. Add 500
μ
L of distilled water as con-
trol. Incubate the samples with the protein reagent at room
μ
