Biology Reference
In-Depth Information
3.2 Protein
Extraction
( See Note 3 )
1. Grind acorn fl our (10 g) in liquid nitrogen with a precooled
mortar and pestle until a fi ne powder is formed (
see
Note 4
).
2. Add 60 mL of diethylether and shake the mixture for 1 h at
4 °C (
see
Note 5
).
3. Centrifuge at 15,000 ×
g
for 15 min (4 °C) and decant the
supernatant (
see
Note 6
).
4. Repeat
steps 2
and
3
two more times.
5. Add 60 mL of acetone containing 0.07 % (w/v) DTT to the
above pellet, and shake the mixture for 10 min at 4 °C (
see
Note 7
).
6. Centrifuge at 15,000 ×
g
for 15 min (4 °C) and decant the
supernatant (
see
Note 8
).
7. Allow the pellet to dry at room temperature until no traces of
acetone remained.
3.2.1 Sample
Delipidation
1. Add 50 mL of ES1 and 1 g of PVPP to the pellet obtained in
the previous step.
2. Sonicate the mixture (while keeping on ice) for 15 s (50 W,
amplitude 60), and shake in an orbital shaker for 2 h at 4 °C.
3. Centrifuge at 15,000 ×
g
for 15 min (4 °C) and transfer the
supernatant to a new tube (
see
Note 9
).
4. Precipitate proteins in the supernatant by adding four volumes
(approximately 100 mL for each tube) of 0.07 % (w/v) DTT
containing cold acetone, and keep 1 h at 20 °C.
5. Centrifuge at 15,800 ×
g
for 15 min (4 °C) and decant the
supernatant.
6. Dry the pellet at room temperature and store it at −80 °C if
not immediately used (
see
Notes 10
and
11
).
3.2.2 Solubilization
of the Albumin Fraction
1. Add 50 mL of ES2 to the pellet obtained in
step 3
(Subheading
3.2.2
).
2. Sonicate the mixture (while keeping on ice) for 15 s (50 W,
amplitude 60), and shake in an orbital shaker for 2 h at 4 °C.
3. Centrifuge at 15,000 ×
g
for 15 min (4 °C) and transfer the
supernatant to a new tube (
see
Note 9
).
4. Repeat
steps 4
-
6
of the albumin fraction solubilization to
obtain the globulin fraction (
see
Note 12
).
3.2.3 Solubilization
of the Globulin Fraction
1. Add 50 mL of ES3 to the pellet obtained in
step 3
(Subheading
3.2.3
).
2. Sonicate the mixture (while keeping on ice) for 15 s (50 W,
amplitude 60), and shake in an orbital shaker for 2 h at 4 °C.
3.2.4 Solubilization
of the Prolamin Fraction