Biology Reference
In-Depth Information
Table 1
Example of differential labeling, mixing, and gel loading for
comparison of thiol reactivity and protein expression under
four treatment conditions in triplicate using cysteine-labeling
2D DIGE
ICy3 (
m
g pool)
ICy5
Gel 1
150
150 mg condition 1 (0 min H
2
O
2
)
Gel 2
150
150 mg condition 1 (0 min H
2
O
2
)
Gel 3
150
150 mg condition 1 (0 min H
2
O
2
)
Gel 4
150
150 mg condition 2 (2 min H
2
O
2
)
Gel 5
150
150 mg condition 2 (2 min H
2
O
2
)
Gel 6
150
150 mg condition 2 (2 min H
2
O
2
)
Gel 7
150
150 mg condition 3 (20 min H
2
O
2
)
Gel 8
150
150 mg condition 3 (20 min H
2
O
2
)
Gel 9
150
150 mg condition 3 (20 min H
2
O
2
)
Gel 10
150
150 mg condition 4 (240 min H
2
O
2
)
Gel 11
150
150 mg condition 4 (240 min H
2
O
2
)
Gel 12
150
150 mg condition 4 (240 min H
2
O
2
)
bromophenol blue solution added. The mixed samples are
allocated appropriately for separation on triplicate 2D-gels as
shown in Table
1
. This scheme controls for dye bias, although
labeling combinations are interchangeable so long as each gel
is loaded with samples labeled with distinct dyes. This experi-
ment generates 24 images for matching, cross-comparison,
and statistical analysis in the biological variation analysis (BVA)
module of the DeCyder software.
7.
Rehydrate Immobiline DryStrip pH 3-10 NL gel strips with
samples for at least 12 h in the dark at room temperature in a
rehydration tray (passive rehydration method). Strips should
be covered with mineral oil.
3.4. Preparation
of 2D-Gels, Imaging,
and Image Analysis
1.
For SDS-PAGE casting, an Ettan DALT
twelve
system (24 cm)
is employed.
2.
Prior to gel casting, treat low-fl uorescence glass plates with
1 mL of fresh Bind-Silane solution per plate, wiping over one
surface with a lint-free tissue. Leave plates to dry for a minimum
of 1.5 h (see Note 7 and Fig.
3
).
3. Treat the clean and dry inner surface of the other plate with 1 mL
Repel-Silane to ensure easy separation after electrophoresis.
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