Proteomic Profiling of the Epithelial-Mesenchymal Transition Using 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 281

18. Carefully slide each strip gel into a separate glass cassette and
slide the strip gel down to the top of the polyacrylamide gel
using a spacer/spatula, so the gels sit fl ush together. Avoid
trapping bubbles between the gels.
Prepare the DALT twelve separation unit for the second-
dimension SDS-PAGE.
19. Fill the lower buffer tank with SDS running buffer.
20. Switch the control unit and pump on and set the temperature
to 23°C.

21. Insert the loaded cassettes into the tank and insert blank

cassettes into any free slots (see Note 18).
22. Fill the upper buffer tank with SDS running buffer.
23. Close the lid and commence electrophoresis. The following
parameters have been successfully used:
(a) 5 W per gel for 30 min.
(b) 90 V at 23°C until the dye front reaches the bottom of the
cassette.

24. Once electrophoresis is complete, scan immediately (see

Note 19).

1. Initialize scanner and prepare for imaging. 3.5. Gel Scanning

and Image Acquisition

2. Dry cassettes with Kimwipes and ensure the complete removal

of fi ngerprints and dust.

3. Place gel into the scanner and set scanning parameters:

(a) Set the acquisition mode to fl uorescence.
(b) Set excitation/emission wavelengths for Cy2 (488/520 nm),
Cy3 (532/580 nm), and Cy5 (633/670 nm).
(c) Set the PMT voltage.
(d) Select the orientation of the gel.
(e) Select to press sample.
(f) Select focal plane to +3 mm.

4. Defi ne the area to be scanned (i.e., gel boundaries).

5. Perform a pre-scan with pixel size set to 1,000 μ m resolution

(see Note 20).

6. Perform fi nal scan at 100 μ m resolution (see Fig. 2 ).

7. Scan all gels.

3.6. Image Analysis
and Protein
Quantifi cation

The images can now be processed by software such as DeCyder 2D
to assist detection, quantitation, matching, and analysis of DIGE
gels. A full description of the software is beyond the scope of the
chapter; however, an extensive and a very useful DeCyder manual
can be obtained from the GE Healthcare website ( http://www.
gelifesciences.com/aptrix/upp00919.nsf/Content/65B08B3DA