Biology Reference
In-Depth Information
Fig.
1
(
a
) Representative 1-D gel of mature olive pollen proteins. Approximately 25
g
of pollen proteins were loaded on 12 % (w/v) polyacrylamide gels with 4 % stacking
gels and proteins were electrophoresed using a Mini-Protean 3 apparatus (catalog
number: 165-3301, Bio-Rad Corp., Hercules, CA, USA) as previously described [
18
].
Proteins were stained with BioSafe Coomassie Stain (catalog number: 161-0786, Bio-
Rad Corp., Hercules, CA, USA) following the manufacturer's instructions. Protein markers
(catalog number: 161-0373, Bio-Rad Corp.) are displayed on the
left
. (
b
) Representative
2-D gel of the mature olive pollen proteins containing 1,021 spots. The global protein
profi le was largely reproducible among replicas. Approximately 100
μ
g of total protein
was loaded on 18 cm polyacrylamide strips, pH 3-10 NL (catalog number: 163-2033,
Bio-Rad Corp.) by in-gel rehydration at 30 V for 12 h. IEF was carried out at 20 °C in a
Protean IEF Cell (catalog number: 165-4000, Bio-Rad Corp.) as follows: 300 and 1,000 V
for 1 h each followed by a linear increase from 1,000 to 10,000 V, and fi nally 10,000 V
to give a total of 40 kVh. Reduction and alkylation steps were performed as previously
described [
19
]. Protein separation in the second dimension was carried out by SDS-
PAGE in a Protean II xi Cell (catalog number: 165-1951, Bio-Rad Corp.) as previously
described [
20
]. Proteins were stained with BioSafe Coomassie Stain (Bio-Rad Corp.)
following the manufacturer's instructions. 2-D SDS-PAGE standards (catalog number:
161-0320, Bio-Rad Corp.) are displayed on the
left
μ
2.2 Protein Extract
Filtration
1. PD-10 Desalting Columns (catalog number: 17-0851-01, GE
Healthcare Biosciences AB, Uppsala, Sweden) or similar (
see
Note 6
).
2. Equilibration buffer: 0.05 M Tris-HCl, pH 7.4.
3. Elution buffer: 0.01 M Tris-HCl, pH 7.4.
2.3 Protein
Precipitation
1. Precipitation solution: 20 % (v/v) trichloroacetic acid (TCA)
and 0.2 % (w/v) DTT in chilled (−20 °C) acetone (
see
Note 7
).
2. Washing solution: 0.2 % (w/v) DTT prepared in chilled
(−20 °C) acetone.
3. Refrigerated centrifuge Sorvall RC-6 Plus (catalog number:
46910, Thermo Fisher Scientifi c Inc., Waltham, MA, USA) or
similar, equipped with a rotor for 50 mL centrifuge tubes.
4. Oak ridge centrifuge tubes (50 mL), polypropylene copoly-
mer (catalog number: 3119-0050, NALGENE Labware,
Thermo Fisher Scientifi c Inc.).
