Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 383

Table 2
Gel solutions to cast three gradient gels ( T = 10-15%,
C = 2.7%) with the equipment described in Subheading 2.4

Gel composition

10% 15%

T30C2.7 18.711 mL 28.083 mL

Separation gel buffer 14.355 mL 14.355 mL

Water 23.1 mL -

70% Glycerol - 13.728 mL

10% Ammonium persulfate 122 mL 122 mL

TEMED 16.5 mL 16.5 mL

Table 3
Composition of stacking gel ( T = 5%, C = 2.7%)

Per gel

T30C2.7 0.683 mL

Stacking gel buffer 1.035 mL

Water 2.427 mL

10% Ammonium persulfate 40 mL

TEMED 5.5 mL

3. Fix the separation gel assembly with the long clamps and put it

upright in the dual gel casting stand. The polymerized gel
should be about 2-3 cm below the upper edge of the glass
plates. Decant any water on top and fi ll the residual space
almost completely with stacking gel solution (for composition
see Table 3 ). Insert the comb and leave to polymerize for about
30 min. The gel is then ready for inserting the equilibrated
IPG strip (see Subheadings 3.5 and 3.6 ).

Equilibration solutions are freshly prepared and aliquots transferred
into the glass tubes. The IPG strips are taken out of the freezer, the
protruding anodal part of the support fi lm is cut off, and the cath-
odal one is only shortened so that it can still easily be held with
forceps. When half-thawed, the protecting transparent covers are
peeled off, each strip put into one glass tube with equilibration
solution 1 and put on a horizontal shaker at 100 rpm for 10 min
(see Note 40). Afterward, strips are transferred to a fresh tube with

3.5. Equilibration