Comparative 2D DIGE Analysis of the Depleted Serum Proteome for Biomarker Discovery - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 216

Table 2
Second-dimension electrophoresis program on the Ettan DALT12

Phase Current (mA)/gel Power (W)/gel Voltage (V) Duration (h)

1 5 0.5 50 2

2 10 0.5 100 1

3 30 2.5 250 14

Fig. 1. A representative 2DE gel of a CyDye-labeled “fl ow-through” fraction following MARS depletion of mouse serum.

The gel should still attach to the glass plate. Hold the edges of
the plastic backing and start pulling the gel away from the glass
plate. Cover the gel with the plastic coversheet to reduce expo-
sure to environmental contaminants (see Note 8). Attach fl uo-
rescent references markers to the central left and right sides of
the fi lm support, approximately 1 cm inside each margin.

15. Scanning should be performed immediately using an Ettan

DIGE Imager (GE Healthcare) at 100 mm resolution. Appropriate
exposure settings for the precast gels are 0.4 s for the Cy2 chan-
nel, 0.07 s for the Cy3 channel, and 0.1 s for the Cy5 channel.
Protect those gels not currently being scanned from light. Once
scanned, replace the plastic coversheet, place the gel in an indi-
vidual ziplock bag, then store at −80°C (see Note 9). Fixation of
the gels is not required unless performing a subsequent staining
procedure. An example gel is presented in Fig. 1 .