Biology Reference
In-Depth Information
equilibration solution 2. After 5 min horizontal shaking, the strips
are recovered and immediately transferred to the stacking gel of
the second dimensional gel.
3.6. Second
Dimension/SDS-PAGE
Take the equilibrated IPG strip out of the glass tube, thereby holding
the cathodal plastic with forceps (see Note 41). Place the strip into
the trough of the stacking gel with the anode to the left and the
plastic backing of the strip toward the back glass plate of the gel
assembly. Make sure that the backing is sticking to this glass plate
and that there is some room between the gel side of the strip and
the front glass plate. This space is now fi lled with warm agarose
(about 700 mL per gel), which helps to keep the strip in place.
Carefully avoid bubbles as they would prevent an even current. Fill
the rest of the gel cassette with running buffer, fi x the upper cath-
ode chamber onto the two gels (or one gel and the place holder),
and transfer the completed assembly into the anodal buffer tank.
Fill the cathodic compartment with running buffer, making sure
that the chamber does not leak (see Note 42). Start the cooling
circulation (set to 15°C). Apply standards and references in the left
and right pockets of the stacking gel, respectively, and start the run
(25 mA constant current per gel). It will take about 4-5 h until the
bromophenol blue reaches the bottom of the separation gel.
1. Switch on the scanner about 20 min before the run is fi nished.
When the bromophenol blue has reached the end of the gel,
stop electrophoresis, remove the gels (gels and glass plates)
from the tank, and rinse the assembly carefully with warm tap
water. Then dry them and put the gels (still between the glass
plates) in the scanner. The scanner is operated according to the
manufacturer's protocol. Scanner settings for a fi rst measurement
should be around 450 V PMT for each channel and 100-mm
pixel size (for a fi rst pre-scan you may select a lower resolution
setting, i.e., larger pixel size). Make sure to select the appropri-
ate focal plane (“+3 mm”). The albumin spot should not be
saturated, otherwise re-scan (see Notes 43 and 44).
2. Further processing depends on the experiment:
(a) Qualitative analysis: The three channels of the false-color
representation of the original scans (red, green, blue) can
be overlaid and—depending on the protein distribution—
result in mixed colors (for a complete overlap of spots and
equal amounts of proteins, this will be white). The images
may be looked at singly or in combination (in false colors),
adjusted in intensity, and also converted to gray scales.
(b) Semiquantitative analysis: This may be done with the
software module DIA (Differential In-gel Analysis) in
DeCyder, which allows pairwise comparison of two chan-
nels. Spots can be detected, fi ltered (according to size,
3.7. Scanning
and Post-detection
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