Analysis of Protein Posttranslational Modifications Using DIGE-Based Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 138

Fig. 2. Orientation and alignment of the IPG strip. Align the strip so that the line to the right of the positive sign overlaps the
electrode as shown.

Step 2: step to 500 V for 30 min.
Step 3: step to 1,000 V for 30 min.
Step 4: step to 5,000 V for 2 h (see Note 19).
13 - cm strips :
Step 1: 30 V for 12 h.
Step 2: step to 500 V for 1 h.
Step 3: step to 1,000 V for 1 h.
Step 4: step to 8,000 V up to 28,000 Vh (see Note 20).
24 - cm strips :
Step 1: 30 V up to 450 Vh.
Step 2: step to 500 V up to 500 Vh.
Step 3: step to 1,000 V up to 1,000 Vh.
Step 4: step to 8,000 V up to 64,000 Vh.
Step 5: 500 V for 24 h (see Note 21).

Use clean tubes when equilibrating IPG strips. For 7-cm strips,
conical 15-mL tubes are useful; for other strips, alternative equili-
bration tubes are available (e.g., from GE Healthcare):

3.5. SDS-PAGE
(Second Dimension)

1. Prepare fresh equilibration buffer approximately 30 min before
use, followed by fresh DTT-EB and Iodo-EB as required.
2. Prepare 1× running buffer for washing the IPG strips; usually
1-2 L, the remainder will be used when running the gel(s).
3. Remove the strip from the holder using forceps and wipe the
back of the strip by dragging it across a folded Kimwipe. Rinse
the strip in 1× running buffer.