Protein Protocols Protein Protocols

Casting IPGs 177
6. Start the mixing on the stirrer at high speed. Slowly move the gradient mixer around the
active area of the mixer in order to find a position in which the magnetic bars in both chambers
turn evenly and synchronously. Reduce the stirring speed.
7. Add ammonium persulfate solution in the stated amounts (see Table 2) to the solutions in
both chambers.
8. Open the outlet to the polymerization chamber; the liquid should easily flow by gravity: in
case it does not, a light pressure (the palm of your hand over the mouth of the vessel)
might solve the problem.
9. When the heavy solution is about to enter the polymerization cassette, open the connection
between the two chambers of the mixer.
10. The in-flowing light solution should mix promptly with its heavy counterpart; if not
(streams of liquid of different density are distinctly seen in the mixing chamber), the stirring
speed should be increased accordingly (see Note 6).
11. When all solution has flown by gravity, force the few drops remaining in the outlet
tubing and in the mixing chamber to the cassette by applying pressure (see above)
first on the reservoir and then on the mixing chamber, while raising the gradient mixer.
12. Inspect the liquid within the cassette: no air bubbles should be present. It is possible to
remove them, with great caution, with the help of a thin hook cut from GelBond foil.
13. Leave the gradient to stabilize for 5–10 min at room temperature before moving the cassette
into the oven to start the polymerization step.
14. After use, promptly fill the gradient mixer with distilled water and flow the water through
the outlet tubing; repeat this procedure twice.
15. Set aside the stirring bars; disassemble the outlet tubing and dry by suction with a pump;
and blow the liquid still remaining inside the gradient mixer with compressed air, before
drying the outside with a paper tissue.
16. Let the equipment dry to completion with all connections in the open position. Rinsing and
drying the gradient mixer, and pouring a new gradient takes ~10 min, so no delay is actually
required when processing a number of IPGs one after another.
3.5. Polymerization Conditions
1. Let the IPGs polymerize for 1 h at 50°C (see Note 7).
2. Gel setting occurs within approx 10 min, whereas polymerization slowly proceeds to plateau
in the next 50 min. It is critical during the early period that the polymerization cassette
is kept in an upright position on a level surface. Moreover, the oven should not be
opened in order to avoid both shaking the cassette and causing the temperature to drop.
3.6. Washing, Drying, and Storing IPGs
1. At the end of the polymerization period, disassemble the cassette by removing the clamps.
2. Insert a scalpel blade between GelBond and glass plate to loosen the foil from its support.
3. Fill a plastic box in which the gel may lay flat with 1 L of glycerol washing solution.
4. Gently peel the IPG from the mold, and transfer (upside up!) into the box containing the
glycerol solution.
5. Gently shake for 1 h at room temperature.
6. Lay the gel (upside up!) on a supporting glass plate partially or completely submerged in the
washing solution in order to avoid trapping air bubbles (see Note 8).
7. Set the assembly upright, approx 30 cm in front of a (cool) fan, operating at high speed. The
whole area should be as dustless as possible; glycerol smudge is better collected on a tray.
8. Evaluate the progression of the drying by sensing the cooling from water evaporation on
the back of the glass plate and by looking for an even and matt appearance on the front of