Method for Protein Subfractionation of Cardiovascular Tissues Before DIGE Analysis - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 294

3. Supplement the deglycosylation buffer with chondroitinase

ABC and keratanase as described above.

4. Incubate samples for 16 h at 37°C.

1. Prior to the DIGE analysis, SDS has to be removed by protein

precipitation (see Note 5). We use a commercial 2DE clean-up
kit (Bio-Rad, Hercules, CA). Resuspend protein pellets in
DIGE lysis buffer.

3.5. DIGE Analysis
of SDS Extracts (see
Note 6 and Fig. 2 )

2. The labeling reaction with CyDye DIGE fl uor minimal dyes is

performed according to the manufacturer's instruction (GE
Healthcare). However, a lower dye-to-protein ratio (200 pmol
dye/50

g protein) is suffi cient because the SDS extracts are
already depleted of high-abundant plasma and ECM proteins.

μ

3. The labeled samples are mixed with the same volume of DIGE

2× buffer. Top up to a fi nal volume of 450 L with rehydration

buffer. Vortex and centrifuge at 16,000 × g for 1 min.

μ

4. Rehydrate Immobiline DryStrip gels (IPG, GE Healthcare) in

a reswelling tray according to the manufacturer's instruction.

Fig. 2. DIGE image of SDS extracts from murine hearts (pH 3-10NL, 18-cm IPG strip,
12% gel).