2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 98

Table 2
Parameters recommended by GE Healthcare for isoelectric
focusing of analytical saturation DIGE samples

Step Power (V) Ramp Duration (h)

1 300 Step and hold 3.0

2 600 Gradient 1.5

3 1,000 Gradient 2.0

4 8,000 Gradient 2.5

5 8,000 Step and hold 3.0

6 500 Step and hold Up to 48.0

Current should be below 25 μ A/strip, and temperature should be adjusted

to 25°C

(e.g., from the Typhoon series; GE Healthcare) usually provide
better resolution and signal-to-noise ratios than camera-based
imaging instruments. Generate overlay images of corresponding
readouts using appropriate software tools (e.g., ImageQuant).

3.4. Evaluation
of the Optimization
Experiment

To decide which labeling condition gives the best labeling results,
carefully inspect the overlay images with respect to the following
parameters:

1. All spots should be overlaid between the Cy3 and Cy5 image;

nonoverlaid spots are an indication for insuffi cient amounts of
CyDye.

2. No signifi cant horizontal spot “arrays” should be present: hori-

zontal spot arrays are an indication for overlabeling. They can
be distinguished from natural protein isoforms differing in their
pI by their disappearance at lower CyDye concentrations.

3. No signifi cant vertical spot “arrays” should be present: vertical

spot arrays are an indication for under-labeling. Labeling-
induced vertical spot arrays can be distinguished from natural
proteins of identical pH but slightly different molecular mass
by their disappearance at higher CyDye concentrations. Small
proteins are more suitable for detection of under-labeling,
since the CyDye dependant differences in molecular mass can
be resolved on the gel in the 10-30 kDa range. Figure 3 shows
an example of under-labeling and the disappearance of under-
labeled spots upon an increase in CyDye amount.

4. Usually, this optimization experiment will lead to at least one

gel without the obstructions mentioned above. If this is not
the case, carefully check all solutions and buffers and the sample
preparation procedure. Cross-check your protein concentration