Biology Reference
In-Depth Information
3.5. Two-Dimensional
Electrophoresis
1.
These instructions are modifi ed from the 2DE technique as
described by Klose and Kobalz (
7
). This technique is based on
IEF employing carrier ampholyte tube gels. It can be easily
adapted to the DIGE technique and other formats, including
analytical as well as preparative gels. In spite of the fact that
most of the instruments were constructed in-house, equivalent
equipment is commercially available. For the second dimen-
sion, the Desaga VA300 gel system is applied.
2.
Two days before running, IEF tube gels (Ø 1.5 mm, 20 cm)
are prepared. Add 45
L of the 1.2% APS solution to 2 mL of
the separation gel buffer and fi ll the tube to the fi rst mark
(20 cm) using a syringe. Now, add 14
μ
L of the 1.2% APS
solution to 0.7 mL of the cap gel buffer and cast the cap gel to
the second mark (20.5 cm) behind the separation gel. To pre-
vent urea crystallization, place an air cushion under the cap gel
to the third mark (21 cm).
μ
3.
Before starting IEF, apply 2 mm of the sephadex solution to
prevent protein precipitation onto the separation gel. Then,
load the sample (dye-labeled) and overlay the sample with
protection solution (approximately 5 mm) to prevent direct
contact of the acidic cathodic buffer (see Note 8).
4.
Fill anodic (bottom) and cathodic buffer (top) into the IEF
chambers. Ensure that no air bubbles hamper IEF. Start IEF
applying a stepwise voltage program (100 V for 1 h, 200 V/1 h,
400 V/17.5 h, 650 V/1 h, 1,000 V/30 min, 1,500 V/10 min,
2,000 V/5 min).
5.
While the 21.5-h IEF is running, gels for the second dimension
should be prepared. Clean the glass plates using a lint-free
paper towel. For the fi rst wash, use double-distilled water
followed by 100% ethanol and fi nally 70% ethanol. To ensure
correct gel dimensions, two 1.5-mm plastic spacers are placed
between two plates sealed by silicon.
6. Add 288
L of the 40% APS solution into 144 mL of separation gel
buffer, cast the gel, and overlay with water-saturated isobutanol.
μ
7.
After polymerization (45 min), remove the isobutanol and wash
the surface with protection solution. To protect the gel from
drying, place two-dimensional polyacrylamide gel electropho-
resis (2D PAGE) protection solution (see Subheading
2.4
)
onto the gel and store the gel at 4°C.
8.
After IEF, extrude the gel by means of inserting a nylon fi ber
(see Note 9) into the gel groove of the IEF gel carrier and
incubate with equilibration solution for 15 min to load
proteins with SDS.
9.
Wash the gel 3 times with 2D PAGE running buffer before
applying to the second dimension.
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