Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 378

gradient maker

gel
cassette

pump

stirrer

Fig. 1. Setup to cast gradient gels for IPG gel plates.

is made and used as an internal standard throughout the whole
study (labeled with Cy2). In the latter case, samples are only
labeled with Cy3 and Cy5. Dye swapping (reverse labeling,
i.e., labeling aliquots of the same specimen with different dyes)
is recommended to avoid bias from preferential labeling.
2. Serum is diluted 1:6 with DIGE labeling buffer and precooled
in an ice bath (see Notes 13 and 14); 2.5 mL of this pre-diluted
sample is mixed with 0.5 mL of the CyDye working solution
(see Note 15) and incubated in the ice bath for 30 min in the
dark. The reaction is stopped by addition of 0.5 mL of the lysine
solution and vigorous mixing. After further 10 min in the ice
bath under light protection, 7 mL of the IPG sample solution is
added. The sample is mixed and after 10 min cooling (in the ice
bath or fridge) ready for immediate use. Alternatively, it can then
be frozen and used at a later time-point (see Notes 16-19).

1. The supporting fi lm is applied to the glass plate of the casting

cassette with the help of a roller (see Note 20). The carefully
cleaned glass plate with the rubber U-frame is wiped with a
paper towel moistened with some milliliters of Repel Silane,
left to dry, and afterward rinsed with plenty of water. After
drying it, it is put on the glass plate with the fi lm and the casting
cassette is fully assembled with the clamps (see Note 21).

3.2. IPG Preparation

2. The casting setup is arranged as shown in Fig. 1 : The gradient

maker with a small magnetic bar in the front chamber is put on
the magnet stirrer and its outlet connected to the peristaltic
pump (see Note 22). The tubing outlet of the pump is put into
the middle notch of the assembled casting cassette (e.g., via a
butterfl y needle or pipette tip).

3. The two casting solutions are prepared in glass vials or tubes

according to the composition given in Table 1 . After addition
of the polymerizing agents, they are immediately fi lled into the
respective compartments of the gradient maker. The alkaline
solution is put into the back compartment. The valve between
back and front chamber is carefully fi lled with this solution
avoiding trapping air bubbles that would hamper liquid fl ow