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Fig. 2. The 2D DIGE image patterns of six narrow pH ranges and the position of representative differentially expressed
spots with a fold change of >±2. Six images (
a
) were combined into one image. (
b
) The “normal” sample was labeled with
Cy3 (
green
) and the “HCC” sample with Cy5 (
red
). Forty-three spots were identifi ed from preparative gels by nanoLC-
MS/MS (
c
) .
analysis (DIA) and biological variation analysis (BVA) mode
(see Note 11).
2.
Using a master gel, match and merge accurately the spots of
the other gels, if necessary. Accept statistically signifi cant spots
(
p <
0.05), and fi lter over the average volume ratio of ±2. Select
and check for accuracy across fi ltered spots of the 2D DIGE
and preparative gels (Fig.
3
) (see Note 12).
3.
Pick each protein spot of interest with an autoclaved end-cut
yellow tip (~2 mm), and transfer the gel piece into a fresh 1.5-
mL tube containing 1 mL of distilled water. Wash the gel piece
twice by adding 100
L of spot destaining buffer (40% (v/v)
50 mM NH
4
HCO
3
in acetonitrile), shaking for 10 min and
discarding the destaining buffer. Repeat this step until the
Coomassie Blue G-250 dye disappears (~5 times). Add 50
μ
L
of 100% acetonitrile, shake for 3 min, and discard the ace-
tonitrile. Repeat this step until the gel piece turns white
μ
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