The Basics of 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 12

Traditional 2D gel electrophores is (Silver, Coomassie, SYPRO Ruby,
Deep Purple)

Need gel replicates and biological replicates.
Minimum needed for statistics is 3 of each.
No. of gels (N) = no. of samples (y) x 3 (biological reps.) x 3 (gel reps.)
N = y x 3 x 3
Example 1: control versus treated (y = 2);
N =2 x 3 x 3 = 18 gels.
Example 2: control versus treated versus treated+drug (y=3);
N =3 x 3 x 3 = 27 gels.

2D DIGE (Minimal Labeling)

Only need biological replicates (since gel-to-gel variation is virtually
eliminated with this technique).
Minimum needed for statistics is 3 - but to properly enable reverse labeling 4
is recommended. The multiplexing capability allows three samples per gel
(one of these is the internal standard so two real samples are run per gel).
No. of gels (N) = no. of samples (y) x 4 (biological reps.) N = y x 4
2 (since two samples on each gel) 2

Example 1: control versus treated (y = 2);
N = 2 x 4 = 4 gels.
2
Example 2: control versus treated versus treated+drug (y=3);
N = 3 x 4 = 6 gels.
2

Fig. 2. Calculation for the minimum number of gels to be run for a traditional 2D experi-
ment compared to a 2D DIGE experiment.

It is important with this technique that the three fl uorescent
dyes (Cy2, Cy3, and Cy5) are imaged with an appropriate device
that can not only independently excite these fl uors but is also able
to distinguish between the three resulting spectra and avoid any
cross talk issues, which would interfere with quantifi cation. A laser
scanner capable of blue, green, and red excitation and equipped
with the appropriate band pass fi lters for the corresponding emis-
sion is highly recommended. Also, a suitably designed image analy-
sis software package should be used to perform the required
calculations ( 7 ). In particular, the software's ability to properly
handle the codetection of the images within each gel, and the nor-
malization against the internal standard, will infl uence the accuracy
and reliability of the quantifi cation.
Since the inception of 2D DIGE in 1997, there are now over
2,500 papers (as of May 2011, Ishida Y, GE Healthcare, personal
communication). Many types of samples have been investigated
using this technique, including a wide range of plant and animal
species ( 7 ). Recently, advances have been made in furthering the
utility of the technique by exploring niche applications. Such
examples include cell surface labeling (

10-12 ), reduced vs. nonre-
duced states ( 13 ), host cell protein monitoring ( 14 ), and samples
from laser microdissection ( 15 ), to name but a few.