Native DIGE of Fluorescent Plant Protein Complexes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 347

Table 1
Solutions for the preparation of a native gradient PAGE gel

12% 6%

Acrylamide (37.5/1) 4.20 mL 2.10 mL

6× Gel buffer 1.75 mL 1.75 mL

Glycerol 1.95 g -

ddH 2 O 3.00 mL 6.65 mL

S 10.50 mL 10.50 mL

4. Mix both solutions well by stirring them on a magnetic stirrer.

5. Place the gradient mixer on the magnetic stirrer and connect

the pipette tip with the casting stand. The gradient mixer has
to show a moderate incline towards the casting stand to achieve
a directed fl ow of the gel solutions.

6. The tube which connects the gradient mixer with the glass

plates is fi xed in the middle of the assembled glass plates by
tape. The tube ends in a cut yellow pipette tip.
7. Start the magnetic stirrer. Chamber 1 has to be stirred properly,
whereas stirring of chamber 2 is not necessary. The stirring
bar in chamber 2 acts as balancer for the solution levels in
both chambers.

8. Add 5 mL of TEMED and 20 mL of the APS solution to both

chambers and mix the solutions gently.

9. First, open the valve between the two chambers to remove

the air in the valve between both chambers. Afterward, open
the front valve. Let the solutions run between the glass
plates.
10. Overlay the cast gel with water-saturated isobutanol. The gel
should polymerize within 90 min.
11. Rinse the gradient mixer immediately with distilled water to pre-
vent the polymerization of residual gel solution in the tubes.

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

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

3.2.2. Stacking Gel

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

paper.

3. Clean a 10-well comb with denatured ethanol (100%) and

insert the dried comb in the glass plate sandwich.

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