Method for Protein Subfractionation of Cardiovascular Tissues Before DIGE Analysis - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 295

Table 1
IEF program for 18-cm IPG strip (3-10 NL)

Step Voltage (V) Duration Vh

Optional 30 Step and hold 12h 360

1 150 Step and hold 2 h 300

2 300 Step and hold 2 h 600

3 600 Step and hold 2 h 1,200

4 1,500 Step and hold 8 h 12,000

5 8,000 Gradient 30 min 2,375

6 8,000 Step and hold 2 h 16,000

Optional 500 Step and hold 2 h 1,000

5. Strips are focused at a maximum of 0.05 mA per IPG strip at

20°C overnight in the dark. An optional desalting step at 30 V
can be added. Table 1 provides an example for an IEF program
suitable for the analysis of cardiovascular tissues using an 18-cm
IPG strip. Once IEF is complete, a constant voltage of 500 V
is applied to prevent protein diffusion.

6. Focused strips are sealed in plastic bags and stored in X-ray fi lm

cassettes at −80°C or processed immediately.

7. To cast large-format polyacrylamide gels (12% T (total acryl-

amide concentration), 2.6% C (degree of cross-linking)), mix
187.5 mL Acrylogel 2.6 (40%) solution with 150 mL of 1.5 M
Tris-HCl, pH 8.8, and 253.5 mL of ddH 2 O.

8. Degas for at least 1 h with continuous stirring.

9. L of TEMED, and 2.4 mL of

10% APS. Stir for about 10 s, then pour the gel solution into
the gel caster. Leave enough space at the top for placing the
IPG strip.
10. Overlay the top surface of the gel solution with overlay solu-
tion (upper phase; see Subheading 2.3 ).
11. Cover the gel caster with cling fi lm.
12.

Add 6 mL of 10% SDS, 252 μ

After 1.5 h, discard the overlay solution, wash briefl y with
ddH 2 O, and overlay with gel storage solution. Leave to
polymerize overnight at RT. If the gels are used at a later
time, seal them with gel storage solution in plastic bags and
keep at 4°C.
13. Equilibrate the IPG strips in equilibration buffer with 1% (w/v)
DTT for 15 min. Shake gently.