Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 387

3.8.2. Applications

The concept of 2D DIGE offers new possibilities for 2DE
applications, especially regarding sensitivity and reproducibility. It
needs only small amounts of samples, is sensitive, and measured
signals are linear over several orders of magnitude. For detection
and quantifi cation, no additional staining step is necessary. Minute
differences in pI and molecular mass between samples may be
measured, and with inclusion of an internal standard, also smaller
variations in spot intensities (correlating to protein concentration)
may reliably be determined compared to conventional stains.
Besides all these major advantages, a few points should be consid-
ered: As with all other methods, protocols need to be carefully
followed (e.g., controlling the pH in the labeling step), and as the
“stain” is based on a different principle (fl uorescent dye labeling of
lysines), results are not always comparable with other detection
methods. Labeling properties of the three different fl uorophores
and possibly interfering compounds present in the sample need to
be considered. Specifi c equipment and fl uorophores are necessary,
making the method expensive. For post-electrophoretic protein
identifi cation, in most cases additional colorimetric staining or
additional equipment (e.g., robotic spot picker) is required.
In the following, some examples illustrate diverse applications
of 2D DIGE in the fi eld of animal proteomics:
(a) Qualitative DIGE: This may be used for simple and rapid

comparison of two or three samples, for instance for detection
of contaminations. All samples are separated on one gel, and
differences are seen at a glance without the need for spot
matching. Figure 3 shows such an example, a check of sample
preparation: Lysates from primary murine bone marrow mac-
rophages that have been kept for a few days in cell culture
medium containing fetal calf serum (FCS) were compared to
the pattern of (dilute) FCS and of (dilute) mouse serum.
Although the cells had been carefully washed before lysis, traces
of highly abundant FCS proteins could be detected ( 14 ). Their
protein spots are seen in violet in the image, resulting from the
color mix of red macrophage and blue FCS protein spots. No
mouse serum proteins were found in traceable amounts (green
spots). Detection of residual proteinaceous medium supple-
ments in specimens derived from cell culture at an early stage
of the experiment may help to optimize sample preparation
and/or to prevent putting these spots on the list of candidates
for further investigation.
(b) Semiquantitative DIGE: Two to three samples may be compared
in a rapid and simple way, even semiquantitatively (if dye-bias
has been excluded, see also Subheading 3.7 , step 2). An example
for isoform detection is given in Fig. 4 , showing a close-up of
a gel with different horse serum samples. Spots in the encircled
area belong to a 1 -antitrypsin (also called Pi). This protein is