Biology Reference
In-Depth Information
DALT
six
system, 5 L of 1× SDS electrophoresis buffer are
required.
4.
Insert the gels with the Immobiline DryStrips in place into the
gel electrophoresis system.
5.
Pour 2× SDS electrophoresis buffer in the top buffer tank to
the fi ll line (1.2 L for the Ettan DALT
six
system).
6.
Run the gel at 2 W/gel for 30 min and then at 5-10 W/gel at
20°C until the bromophenol blue dye front reaches the bottom
of the gel.
Once the gel run is fi nished, gels should immediately be scanned to
avoid the diffusion of protein spots. If they are not immediately
scanned, keep the gels moist between the glass plates in the dark
and at 4°C. However, care should be taken to allow the gels to
reach room temperature before scanning. Do not fi x the gels in gel-
fi x before the gels are scanned, as this may affect the DeCyder
Differential Analysis Software quantitation of the labeled proteins.
3.9. Scanning Gels
and Image Analysis
1.
In the Fluorescence Setup window, select the appropriate emis-
sion fi lter from the emission fi lters list. This list displays the
fi lters that are installed on the Typhoon Variable Mode Imager
along with a description of the typical fi lter use. The scanner
control software automatically suggests the laser to be used
with the emission fi lter selected.
2.
Prescan at 1,000 mm pixel resolution to identify a suitable PMT
voltage. The prescan can be opened in ImageQuant software.
Spots showing the most intense signal should be selected using
one of the Object Tools such as the rectangle.
3.
High-resolution (100 mm) scans must be used to collect quan-
titative data. This resolution is required for subsequent image
analysis using the DeCyder Differential Analysis (DA) Software.
The maximum pixel value should not exceed 100,000 as this
indicates signal saturation has been reached and will prevent
quantitative analysis from being achieved. A target maximum
pixel value of 50,000-80,000 is usually suitable.
4.
Once the voltage has been optimized for one gel in an experi-
ment, these settings should be used for all similar gels within
the same experiment. The maximum pixel value should be
within the specifi ed range for all gels to enable accurate quan-
titation of spot volumes.
The analysis with the Decyder v7.0 software allowed the detec-
tion of 2,111 spots in Fraction F2 (membrane/organelles)—55 of
these with signifi cant variation (19 increased and 36 decreased in a
ratio >2.0 or <−2.0)—and 2,416 spots in Fraction F3 (nuclear)—80
of these with signifi cant variation (51 increased and 29 decreased
in a ratio >1.5 or <−1.5; see Figs.
1
and
2
).
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