Analysis of Protein Posttranslational Modifications Using DIGE-Based Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 137

3.3. Total Protein
Labeling (Minimal
Labeling)

Strip holders should be cleaned before beginning the NHS-Cy dye
labeling procedure (see Note 16). When running 24-cm strips, a
2-h prehydration step should also be included prior to labeling
(see Note 17):

1. To label total proteins in a sample, add 1 μ L of NHS-Cy dye

(400 pmol) per 50 g of sample protein and incubate on ice,

protected from light, for 30 min.

μ

2. Add 1 μ L of 10 mM lysine and incubate on ice for 10 min.

Strip holders should be cleaned before beginning the NHS-Cy dye
labeling procedure in order for the strip holders to have suffi cient
time to dry. Prepare fresh 2× SB and rehydration buffer as required:

3.4. Isoelectric
Focusing (First
Dimension)

1. Combine the labeled samples for each strip. For example, the

oxidation (AlexaFluor 488 C 5 -aminooxyacetamide) and Cy3-
labeled aliquots of total sample plus the Cy5-labeled IC.

2. Add a volume of 2× SB equal to the total volume of the com-

bined samples. Incubate on ice for 15 min.

3. Add rehydration buffer up to a fi nal volume for the strip size

used:
7-cm strip—150

L.
13-cm strip—250

μ

μ L.

24-cm strip—450 μ L (see Note 17).

4. Load the sample into the strip holder being careful to avoid

creating bubbles. Use a pipette tip to spread the sample onto
the bottom of the strip holder, making sure there are no gaps
between the walls and the bottom of the holder. Remove any
bubbles before continuing.

5. Peel the protective layer from the strip while holding the non-

gel end with forceps. Lay the strip gel-side down on top of the
sample. Again, be careful to avoid creating bubbles (see
Note 18). Align the line marked on the positive end of the
strip with the electrode as shown in Fig. 2 .

6. Apply cover fl uid. Add 50-100 μ L at a time. Use a maximum

of 100 L for 24-cm strips.

Apply the cover fl uid by dispensing slowly while dragging the
tip gently along the back of the strip. Add the strip holder
cover.

μ L for 7-cm strips and up to 300 μ

7. Place the strip holders on the IPG unit with the cathode and

anode ends aligned as indicated on the apparatus. Close the lid
carefully and protect samples from light.

8. Use the following settings for different strip sizes:

7 - cm strips :
Rehydration: 14 h at 20°C.
Step 1: 30 V for 30 min.