Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 379

Table 1
Composition of IPG gel for a nonlinear pH 4-10
gradient ( T = 4.0%, C = 4.0%; see Notes 25 and 26)

Solution Acidic ( m L) Basic ( m L)

Acrylamido buffer pKa 1 193 24

Immobiline II pK 4.6 193 -

Immobiline II pK 6.2 228 69

Immobiline II pK 7.0 - 91

Immobiline II pK 8.5 - 34

Immobiline II pK 9.3 - 64

1 M Acetic acid - 21

T30C4 1,015 1,015

70% Glycerol 1,500 -

Water 4,471 6,282

TEMED 4.6 4.0

10% Ammonium persulfate 40 40

later on; it is closed again immediately afterward. Then, the
acidic solution is added in the front (mixing) compartment.
The connecting valve is opened, and at the same time, the
pump (at low speed) and the magnet stirrer are started. The
cassette is thus slowly fi lled with gel solution (see Note 23).
Take care to adjust/lower mixing speed when the solution
volumes decrease. At the very end of the gradient when the
solutions run out, incline the gradient maker to get all remain-
ing liquid droplets into the pump to achieve the complete
desired pH range (see Note 24). When the entire gel solution
is fi lled into the cassette, disconnect the cassette and wash the
gradient maker, pump, and tubing immediately with plenty of
water. The cassette with the gelling solution is put into the—
still cold—dryer/oven and the gradient is allowed to settle for
5-10 min. Overlaying the completed gelling solution with
water is optional. Although it helps forming a good meniscus
and keeping off oxygen, there is the risk of mixing the two layers,
resulting in a “diluted” and softer gel at the cathodic end.
4. After the short settling period, the oven is turned on to 50°C
and the gel allowed to polymerize for 1 h. Thereafter, the gel
cassette is removed from the oven and allowed to cool to room
temperature. Optionally, this can be followed by shortly putting