Biology Reference
In-Depth Information
300 V for 3 h, 1,000 V for 6 h, a gradient step up to 8,000 V
during 3 h, and a constant step at 8,000 V to reach 60,000 V h
with a maximum current setting of 50
A/strip.
8. If the IPG strips are not used directly after the IEF run, they
can be stored at −80 °C between plastic sheets and maintained
horizontally.
μ
3.4 Second-
Dimension Separation:
SDS-PAGE
Gels can be cast in the laboratory or precast gels can be acquired
from different companies (e.g., GE Healthcare, Serva).
1. Casting of the gels between low-fl uorescence glass plates:
The glass plates are placed in the gel caster. The homoge-
neous solution for gels is fi rst cooled (4 °C) and degassed
before addition of APS and TEMED. These last two com-
pounds catalyze the polymerization of the gels and thus can be
added directly only before casting. This solution is poured into
the gel caster, and 1 mL of water-saturated butanol is added on
top of each gel. The polymerization must be performed for at
least 4 h.
3.4.1 Vertical Gels
2. Before the second dimension, the IPG strips are equilibrated in
a buffered solution containing SDS (
see
Note 8
), saturating
the proteins in the IPG strips with negative charges to allow
the mass-dependent separation of the protein in the second-
dimension gel. A fi rst step is carried out in equilibration buffer
containing 1 % DTT (w/v) for 15 min and then a second step
with the equilibration buffer complemented with 2.5 % (w/v)
IAA to avoid formation of random S-S (
see
Note 9
).
3. After equilibration, IPG strips are deposited on top of the
SDS-PAGE and then sealed with hot agarose solution.
4. The gels are put in the electrophoresis vertical tank “Ettan Dalt
twelve” and the running buffer is poured into the tank. The
second-dimension run is started either for several hours (fi rst
30 min at 1.5 W per gel, then 17 W per gel for 5 h) or overnight
(fi rst 30 min at 1.5 W per gel, then 2.5 W per gel for the night)
at 20 °C until the dye front reaches the bottom of the gels.
The HPE™ (high-performance electrophoresis) FlatTop Tower
allows electrophoretic separations of up to four horizontal fl atbed
gels at the same time.
1. Preparing the HPE gels: The electrode wicks from the kit are
soaked for 15 min with electrode buffer, one wick with anode
buffer and one with cathode buffer per gel. 4 mL of cooling
solution is poured on the cooling plate. The gel is then set,
gel side up, and IPG strip-slot into the gel toward the cathode,
on the cooling plate. The cooling solution should form a
homogeneous layer, without air bubble, between the gel and
the plate. Finally, the cathode wick is placed on the gel, with-
3.4.2
Horizontal Gels
