Biology Reference
In-Depth Information
3.4 One-Dimensional
Gel Electrophoresis
Prepare the 13 % polyacrylamide gel electrophoresis using a
PROTEAN II cells (Bio-Rad, Hercules, USA) electrophoresis kit.
1. Prepare the resolving gel solution by mixing 27.08 mL of 30 %
of acrylamide, 15.6 mL of Tris-HCl, pH 8.8, 0.625 mL of
SDS, 18.8 mL of distilled water, 0.31
L
of TEMED. Pour the solution into the gel cassette and cover
completely the solution surface with isopropanol to obtain a
fl at layer on top of the resolving gel. Leave at room tempera-
ture until 20 min.
2. When polymerization is completed, prepare the stacking gel.
Mix 1.63 mL of 30 % acrylamide, 2.5 mL of Tris-HCl, pH 6.8,
0.1 mL of SDS, 6.75 mL distilled water, 50
μ
L of APS and 31.2
μ
L
TEMED and gently stir to obtain a uniform solution. Pour the
resolving gel and transfer the well-forming comb into this
solution. Polymerize the gel for at least 30 min at room tem-
perature to allow complete polymerization.
3. The comb is removed from the stacking gel and place the gel
in the electrophoresis tank. The gel is covered with running
buffer, and 70
μ
L APS and 10
μ
L of sample is applied to the bottom of each
well. The volume and protein concentration of the sample
should be suffi cient to give at least 50
μ
μ
g of each protein. Apply
L of the molecular weight standards to one or two wells,
preferably in an asymmetric position.
4. Connect the wires to the power supply unit and apply 100 V
until the blue dye front reaches the bottom of the gel.
Disconnect the electrophoresis unit from the power supply,
remove the lid and discard the running buffer. Remove the gel
from plates with a spatula, discard the stacking gel, and wash
the separated gel with distilled water to remove traces of run-
ning buffer.
5. Prepare the Coomassie brilliant blue G-250 staining 1 day
before the staining process [
23
]. Place the gel in a tray contain-
ing 500 mL of staining solution. Incubate overnight the gel in
the staining solution. Once the gel is stained, discard the stain-
ing solution and cover the gel with 0.1 M Tris-H
3
PO
4
. Then,
shake for 1-3 min. Discard the solution and cover the gel with
25 % (v/v) of methanol. Then, shake for 1 min. Remove the
methanol and wash the gel with 20 % (w/v) of ammonium
sulfate for 24 h.
6. Images are digitized using a GS-800 Calibrated Densitometer
(Bio-Rad, Hercules, USA) and analyzed with Quantity One
software (Bio-Rad, Hercules, USA).
7. Excise the bands of interest from the gel, and place them in
different tubes containing distilled water until their
processing.
10
μ
