Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 377

2.6. Second
Dimension/SDS-PAGE

1. Running buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS.
2. Sample buffer (for additional standards or controls): Stacking
gel buffer diluted to 1:4 and made up to 3% (w/v) SDS; include
a trace of bromophenol blue.
3. SE600 vertical electrophoresis chamber (Hoefer Scientifi c
Instruments).
4. Refrigerated cooling bath.
5. Power supply for up to 600 V.
6. Agarose: 1% in running buffer (e.g., Indubiose A37). Agarose
stock solution is prepared by dissolving solid agarose in buffer on
a magnetic stirrer at 100-150°C; then a trace of bromophenol
blue is added and the solution stored in aliquots at 4-6°C.
For 2 DE, the appropriate number of aliquots is heated at 95°C
on a Thermomixer 5436 (Eppendorf, Hamburg, Germany).
After complete melting, it is cooled down to 65°C for overlay-
ing the IPG strip after transfer (see Note 8).
7. Standards: molecular weight markers; in the second pocket,
either another marker or a reference sample may be used (see
Note 9). For application of these samples through the running
buffer, use a long form of tips (e.g., Gel-Load; Greiner Bio-One,
Kremsmünster, Austria, see Note 10).

2.7. Scanning
and Post-detection

1. Typhoon 9400 Variable Mode Imager (GE Healthcare).
2. Software DeCyder Version 5.02 and ImageQuant Version 5.2
(both GE Healthcare), or higher.
3. Optional: MS-compatible silver stain, needing the following
solutions (see Note 11):
(a) Fixing solution: 30% ethanol, 10% acetic acid.
(b) Sensitizer: Dissolve 0.5 g sodium thiosulfate (Na 2 S 2 O 3 ·
5H 2 O) and 17 g sodium acetate (CH 3 COONa·3H 2 O) in
175 mL of water, add 75 mL of ethanol.
(c) Silver nitrate: 0.4 g silver nitrate/200 mL.
(d) Developer: Dissolve 18.75 g anhydrous sodium carbonate
(see Note 12) in 750 mL of water and add 75 mL of 37%
formalin.
(e) Stop solution: 1% glycine.

3. Methods

1. Setup of the experiment: For qualitative DIGE, any three sam-
ples can be labeled with the three different dyes. For quantitative
applications, a pool of all samples of the respective sample set

3.1. Sample
Preparation/DIGE
Labeling