Proteomic Profiling of the Epithelial-Mesenchymal Transition Using 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 280

BC1256EAE002E3030&newrel&hidesearchbox=yes&modul
eid=164458 ). Below are brief instructions to cast 8-18% SDS-
PAGE gels:
1. Clean the gel casting cassettes with water and methanol and
assemble cassettes into the gel caster.
2. Prepare light and heavy acrylamide solutions and mix thor-
oughly (see Note 16).
3. Pour the light solution into the right side of the gradient maker
(the chamber which is narrower at the bottom).
4. Allow the light solution to fi ll the tubing and “Y-connector”
between the light and heavy chambers.
5. Close all three pinch clamps on the light and heavy chamber
exit tubes and feed tube.
6. Pour the heavy solution into the left side of the gradient maker
(the chamber is wider at the bottom) until the liquid level
reaches a level 2 cm below the top level of the light solution in
the light chamber.
7. Connect the gel caster to the gradient maker.
8. Add 75 mL of displacing solution into the balance chamber.
Ensure the feed tube is tightly connected to the grommet seal
to prevent leakage of displacing solution into the caster.
9. Open the pinch clamps on the feed tube and light chamber exit
tube.
10. Start the peristaltic pump and open the heavy chamber exit
tube clamp as soon as the light solution level falls from 2 to
1 cm above the level of the heavy solution.
11. Stop the pump when the gradient maker is empty (see Note 17).
12. Pull the feed tube out of the balance chamber grommet and
allow the displacing solution to fl ow into the V-well at the bot-
tom of the gel caster.
13. Spray SDS running buffer immediately onto the top of each
gel in the caster. The level of buffer should be 0.5-1 cm above
the top of the gel.
14. Allow the gel to polymerize at RT for at least 2 h but preferably
for 24 h.
15. Unload the gels from the caster and wash any excess acrylamide
on the outside of each cassette off with water.
16. Store the gels in an airtight container at 4°C with a small
amount of gel storage solution to keep the gels from drying
out, or proceed with strip gel loading.
Place the polyacrylamide gels in the cassette rack and prepare
for strip gel loading and SDS-PAGE.
17. Remove strip gels from the equilibration buffer and rinse in
SDS running buffer.