Native DIGE of Fluorescent Plant Protein Complexes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 350

Table 4
Solutions for the preparation of a SDS
PAGE stacking gel

5%

Acrylamide (37.5/1) 0.8 mL

2× Stacking gel buffer 2.50 mL

ddH 2 O 1.70 mL

S 5.00 mL

3.5. Sample
Preparation for SDS
PAGE Electrophoresis

1. Disassemble the glass plate sandwich of the native gel and
separate the strips of the fi rst-dimension gel by cutting them
with the spacer (see Note 18).
2. Remove the stacking gel.
3. Fill 30 mL of solubilization buffer in a 50-mL centrifuge tube.
4. Transfer the strip in the tube containing the solubilization
buffer by grabbing the native strip at the 12% end.
5. Shake the strip at room temperature for 20 min (see Note 19).
6. After polymerization of the SDS PAGE stacking gel, rinse the
gel with distilled water to remove the isobutanol. Remove the
water on top of the SDS PAGE stacking gel with Whatman
paper.
7. Position the strip between the glass plates of the SDS PAGE
gel touching the surface of the SDS PAGE stacking gel.
Take care that the native and the SDS PAGE stacking
gel touch well. Avoid air bubbles between the first and
second-dimension gel.
8. Overlay the fi rst-dimension strip with overlay solution and let
the agarose thicken.

1. Prepare the SDS PAGE running buffer by diluting 200 mL of
the 10× SDS running buffer with 1,800 mL of distilled water.
2. As soon as the agarose is set, assemble the electrophoresis
apparatus and pour the 1× buffer into the upper and the lower
buffer chamber.
3. Assemble the electrophoresis unit completely and connect to a
power supply.
4. Set the power supply to 5 mA, 600 V and 24 W and start the
electrophoresis for an overnight run (see Note 20).
5. Stop electrophoresis when the bromphenol blue front has
reached the bottom of the separating gel.

3.6. SDS PAGE
Electrophoresis
(Second Dimension)