Native DIGE of Fluorescent Plant Protein Complexes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 349

11. Stop the thermostatic cooler and disassemble the buffer

chamber assembly. The glass plate sandwich can be directly
used for detection of fl uorescence complexes and is scanned in
a fl uorescence scanner for CyDye and pigment fl uorescence.
The use of low fl uorescent glass plates is recommended.
Adjustments for detection of Cy5 are used to scan for pigment
fl uorescence.

3.4. Casting of SDS
PAGE Gels (Second
Dimension)

1. Clean glass plates and spacers with denatured ethanol (100%),

assemble the glass plate sandwich, fi x it on the casting stand
and adjust the assembled casting stand by a spirit level (see
Note 16).

3.4.1. Separating Gel

2. Prepare the gel solution as stated in Table 3 in a beaker and

dissolve the urea by stirring (see Note 17).

3. After dissolving the urea add 15 mL of TEMED and 50 mL of

the APS solution to the solution and pipette the gel solution
between the glass plates. The level of the gel should be approx-
imately 5 cm below the top of the smaller glass plate.

4. Overlay the cast gel with water-saturated isobutanol. The gel

should polymerize within 60 min.

1. After polymerization of the separating gel, remove the isobu-

tanol by washing with distilled water (see Note 10).

3.4.2. Stacking Gel

2. Dry the area above the separating gel completely with Whatman

paper.

3. Prepare the gel solution stated in Table 4 in a beaker, add 5 mL

of TEMED and 50 mL of the APS solution, and pipette the
gel solution between the glass plates. The stacking gel should
be approximately 2 cm high (see Note 14).

4. Overlay the cast gel with water-saturated isobutanol. The gel

should polymerize within 15 min.

Table 3
Solutions for the preparation of a SDS
PAGE separating gel

12.5%

Urea 7.21 g

Acrylamide (37.5/1) 12.5 mL

8× Separating gel buffer 3.75 mL

ddH 2 O 9.00 mL

S 30.00 mL