Biology Reference
In-Depth Information
3.5 Two-Dimensional
Electrophoresis
Preliminary 2-DE experiments can be carried out with the Mini-
Protean 3 system (Bio-Rad, Hercules, USA), using 7 cm pH 3-10
linear gradient strips (Bio-Rad, Hercules, USA) and 13 %
polyacrylamide gels, to examine the p
I
range where the proteins
are concentrated. In our case, the most protein spots were located
in a p
I
range between 5 and 8.
1. Prepare a mix with 300
g of proteins in 250 mL of rehydra-
tion solution on 1.5 mL tube. Load the samples in each lane of
the 17 cm strip holders.
2. Remove the protective cover from the surface of the IPG strips
and slowly lower the IPG strip (gel slide down) onto the rehy-
dration solution, without trapping air bubbles. Then cover the
IPG strip with 1-2 mL of mineral oil and apply the plastic
cover.
3. Apply a low voltage (50 V) during rehydration for 12 h at
20 °C for improving the entry of high molecular weight pro-
teins [
33
].
4. After active rehydration, start isoelectric focusing at 20 °C
using the following parameters 250 V for 2 min, followed by
150 min linear gradient from 250 to 10,000 V, and fi nally
focus on up to 40,000 V at 10,000 Vh.
5. After IEF, the strips are immediately reduced and alkylated
according to [
33
]. IPG strips are equilibrated in two steps.
Firstly, it is performed with 2 % (w/v) DTT in equilibration
buffer I for 10 min in agitation at room temperature; secondly,
it is carried out with 2.5 % (w/v) iodoacetamide in equilibra-
tion buffer II for 10 min in agitation at room temperature.
6. The second dimension is performed on 13 % polyacrylamide
gels using the Protean Dodeca Cell (Bio-Rad, Hercules, USA).
The gels can be run at 150 constant volts until the dye reaches
the bottom of the gel.
7. The gels are stained employing the colloidal Coomassie
method [
23
]. Soak the gel in a tray containing 50 mL of stain-
ing solution. Incubate overnight the gel in the staining solu-
tion. Once the gel is stained, discard the staining solution and
cover the gel with 0.1 M Tris-H
3
PO
4.
Then, shake during
1-3 min. Discard the solution and cover the gel with 25 %
(v/v) of methanol. Then shake during 1 min. Remove the
methanol and wash the gel with 20 % (w/v) of ammonium
sulfate for 24 h.
8. Images are digitized using a GS-800 Calibrated Densitometer
(Bio-Rad, Hercules, USA), and then analyzed with PD-Quest
software v8.1 (Bio-Rad, Hercules, USA).
μ
