Biology Reference
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another hour, and then at 2 W/gel overnight at 24°C in an Ettan
DALT 12 apparatus (GE Healthcare). Next morning, gels were
scanned, and fl uorescently labeled proteins were visualized by the
Typhoon Trio Variable Mode Imager (GE Healthcare) using the
following parameters: Cy2 dye-labeled proteins using 488 nm
excitation wavelength and 520BP40 emission fi lter (520 nm cen-
ter, 40 nm bandpass), Cy3 dye-labeled proteins using 532 nm exci-
tation wavelength and 580BP30 emission fi lter, and Cy5-labeled
proteins using 633 nm excitation and 670BP30 emission fi lter. All
gels were scanned at 100-mm resolution, the PMT (photomulti-
plier tube) voltage was adjusted to keep all the recognized gray-
scale image values below 80,000, which are within the linear range
of the scanner. Images were cropped using ImageQuant v.5.2 soft-
ware (GE Healthcare) to remove areas outside of the gel image.
Image analysis was performed using the DeCyder software v. 6.5
(GE Healthcare). The same software parameters were applied for
the analysis of all images in the automatic batch mode. The esti-
mated number of spots was set in the DeCyder DIA module at
2,500 in all cases. Gel matching was performed within the DeCyder
BVA module automatically without land-marking or manual match-
ing to minimize the operator impact on the analysis. The following
fi ltering criteria were applied for further protein-of-interest (POI)
selection: spot presence in all spot maps, average ratios of ³1.4 and
£−1.4, t test £0.01, and spot volume of ³1.0e + 05 and £1.0e + 07.
3.2.4. Image Analysis
In order to determine how similarly the newly synthesized (LAB)
and commercially available dyes (COM) behave in DIGE, two iden-
tical DIGE experiments were conducted using LAB dyes and COM
dyes. For each experiment, ten protein samples comprising fi ve WT
and fi ve Fis samples were used for labeling ( N = 5). The dyes were
“swapped” to minimize the potential effects of individual dyes: three
samples in the WT group were labeled with Cy3, and two with Cy5,
while three Fis group samples were labeled with Cy5, and two with
Cy3. In both cases (LAB and COM), the Cy2 dye was used to label
the common internal standard consisting of equal aliquots from all
ten samples. Cy3 and Cy5 samples were paired randomly in order to
avoid bias related to loading, and the Cy2-labeled internal standard
was loaded onto each gel along with a Cy3/Cy5 sample pair.
Although experiments with LAB and COM dyes were run indepen-
dently and conducted on two separate days, spot patterns were found
to be remarkably similar (a portion of two gels representing each
experiment is shown in Fig. 4 ). A nonsupervised spot detection was
performed on both sets of gels representing fi ve WT and fi ve Fis
samples labeled with two different dyes. As shown in Table 1 , the
number of detected spots for both LAB- and COM-labeled samples
is very similar across all gels representing both LAB and COM
experiments with the differences of 4.5 and 6% in CV values.
3.2.5. Validation Results
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