2D DIGE Analysis of Protein Extracts from Muscle Tissue - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 162

3.5. IPG Strip
Equilibration

1. Separate the equilibration buffer into two aliquots and just

prior to use add 65 mM DTT (100 mg/10 mL equilibration
buffer) and 135 mM iodoacetamide (250 mg/10 mL equili-
bration buffer), respectively.

2. Equilibrate strips for 15 min in the fi rst solution (DTT)

followed by equilibration in the second solution (iodoacet-
amide) for 8 min.

3. After equilibration, place the strips on fi lter paper moistened

with deionized water.

3.6. Second Dimension
(SDS-PAGE)

A commonly used second dimension gel for 2DE is a homoge-
neous gel containing 12% total acrylamide (see Note 16):

1. Assemble the gel cassette.

2. Prepare a suffi cient volume of gel solution without adding 10%

(w/v) ammonium persulfate (APS) and TEMED.

3. Degas the gel solution for 10-15 min.

4. Add 10% APS solution and TEMED to the gel solution in order

to make a 0.5% (v/v) APS/0.03% TEMED gel solution.

5. Pour the gel solution into the gel cassette and pipette 1-1.5 mL

of 30% (v/v) isopropanol solution on top.

6. Allow gels to polymerize for at least 1-2 h.

7. Disassemble the gel cassette.

8. Prepare the appropriate volume of the 1× running buffer by

diluting the 10× SDS-PAGE running buffer.

9. Melt the agarose sealing solution and, for each Immobiline

DryStrip, slowly pipette the solution up to the top of the glass
plate (see Note 17).

10. Carefully place the Immobiline DryStrip between the two

glass plates holding one end of the strip with forceps. Using a
thin plastic spacer, push the strip until it comes in contact
with the surface of the gel avoiding the trapping of air bubbles
(see Note 18).
11. Insert the glass plates into the electrophoresis apparatus and
start the run (see Note 19).
12. At the end of the run, the gels are ready to be scanned.

3.7. Image Acquisition

After electrophoresis, take the images of the gel at the appropriate
wavelength using a laser-based scanner such as a Typhoon variable
scanner imager (GE Healthcare) ( 11 ) (see Note 20) or alterna-
tively images can be taken using a CCD camera:

1. Rinse the gel sandwich with distilled water and wipe it dry with

lint-free towels.

2. Place the gel sandwich on the scanner platen and take the

images according to the manufacturer's instructions.