Biology Reference
In-Depth Information
using fi ne gel loading tips, until it contacts the acrylamide gel.
Be careful to avoid creating bubbles (see Note 22).
7.
Melt an aliquot of 1% agarose containing bromophenol blue
by microwaving for 5-10 s at a time. Cover the strip by slowly
adding the agarose, cover the plate with aluminum foil, and
allow the agarose to cool.
8.
Load the plate(s) into the electrophoresis chamber as described
by the manufacturer and add the appropriate amount of 1×
running buffer. For large gels, connect the chamber to a circu-
lating water cooler set at 14°C. If using fl uorescent labels,
cover the electrophoresis unit with aluminum foil.
9. Use the following settings for the different strip sizes and gels:
7
-
cm strips
/
10
-
cm gels
.
100 mA for 1 h.
13
-
cm strips
/
gels
.
600 V, 9 mA per gel, 1 W per gel for 16 h.
24-cm strips/gels.
600 V, 25 mA per gel, 1 W per gel for 16 h.
At the end of the run, if the bromophenol blue has not
reached the bottom of the gel, continue the electrophoresis
until it does. Do not exceed 8 W per gel.
3.6. Gel Scanning
and Data Analysis
Gels should be scanned with the appropriate imaging/scanning
equipment for fl uorescent dyes, being careful that the image inten-
sity (PMT Volts) is kept just below saturation levels. Data analysis
can be performed with various software packages. In our laboratory,
we use a Typhoon Trio + (GE Healthcare) imager and the DeCyder
2D 7.0 software. For additional statistical analysis, we have used
Q
-value (
6
) and SPSS.
Two examples of DIGE-based PTM analysis are shown in
Fig.
4
. Panel “a” shows postsynaptic densities isolated from rat
hippocampus that were treated following the protocol described in
Subheading
3.2.3
. As indicated by the arrows, spots shown as
green in the 2D gel correspond to ubiquitinated proteins, which
can be removed from the polyacrylamide gels and identifi ed by
mass spectrometry. Panel “b” displays a portion of the 2D gel in
which dephosphorylated proteins (see Subheading
3.2.1
) were
separated by gel electrophoresis. The absence of the phosphate
group (or groups) induces pI shifts (lower portion of the fi gure)
that can be used to determine the presence of the phosphate group
in the untreated (phosphorylated) sample.
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