2D DIGE Analysis of Protein Extracts from Muscle Tissue - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 160

for the 5 nmol pack size). The stock solution is stable for
3 months at −20°C.

3. Vortex vigorously for 30 s.

4. Centrifuge the vial for 30 s at 12,000 × g in a microcentrifuge.

5. Prepare the CyDye working solution adding 1 volume of CyDye

stock solution to 1.5 volumes of high-grade DMF to make
400 mM CyDye solution. For example, take 2 mL CyDye stock
solution and add 3 mL DMF to give 400 pmol of CyDye in 1 mL.
The CyDye fl uor working solution is stable for 1 week at −20°C.

6. Label 50 mg of each sample with 400 pmol of dye adding 1 mL

of working solution.

7. Carry out the labeling reaction by incubating the sample on ice

for 30 min in the dark.

8. Add 1 mL (for 50 mg of sample labeled) of 10 mM lysine solu-

tion to stop the reaction. Mix by pipetting and spin briefl y in a
microcentrifuge.

9. Leave for 10 min on ice in the dark.

10. After labeling, samples can be stored at −70°C for at least
3 months.

3.3. Loading Samples
onto IPG Strips

1. Add to the CyDye-labeled samples an equal volume of 2× sam-

ple buffer and leave on ice for 10 min.

2. Combine the differentially labeled samples (two or three) into

a single microfuge tube and mix. One of these samples should
be the internal standard (see Note 10).

3. Add to the samples the appropriate volume of rehydration

solution (see Note 11).

4. Pipette the appropriate volume of rehydration solution into

each holder removing any larger bubbles (see Note 12).

5. Remove the protective cover from the IPG strip and position

the strip with the gel side down. Be careful not to trap bubbles
under the IPG strip.

6. Apply the appropriate volume of IPG cover fl uid on the strip

(see Note 13).

7. Allow the IPG strip to rehydrate for a minimum of 10 h

(see Note 14).

3.4. First
Dimension (IEF)

1. Run the appropriate IEF protocol (see Note 15). Figure 3 shows
examples of 2D gels obtained separating labeled samples on dif-
ferent pH gradient. The IEF protocols used are also described.

2. If the IPG strips are not run immediately on the second dimen-

sion, they can be stored at −80°C in a sealed container. Do not
equilibrate strips prior to storage.