The Basics of 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 15

The equilibration solution contains urea, glycerol, reductant, SDS,
and tracking dye. The second equilibration step replaces the reduc-
tant with iodoacetamide to alkylate the reduced cysteine residues.
The strips are then sealed with agarose on to the top of the second
dimension gel and then separated for molecular weight by classical
SDS-PAGE.

If the gel is to be imaged while still between the glass plates or
attached to a plastic backing, then the glass or plastic must have
low fl uorescent properties to minimize any background issues
(autofl uorescence) that could compromise quantifi cation.
The gels are imaged with a suitable fl uorescent imager that is
capable of exciting the three dyes independently and has the neces-
sary band pass fi lters to avoid cross talk (see Note 8).
The image capture is then performed as described in the instru-
ment manual, with the following guidelines:
(a) The fi nal image is scanned at 100-

3.4. Gel Imaging

m resolution.
(b) The fi le format should be a 16 bit .tif (or similar).
(c) Steps should be taken to avoid introducing any fl uorescent
particles (dust, lint, etc.).
(d) The gel images should fi rst be prescanned using a short-
exposure or low-resolution setting so that the fi nal image
capture settings can be optimized to avoid saturation while
taking advantage of the full dynamic range.

μ

Each gel set (three images) should be cropped to remove any areas
that are redundant from the analysis (such as the dye front and IPG
strip) that may interfere with spot detection and normalization.
The cropping should be performed to keep similar spot patterns
the same rather than using similar sized crop areas. The analysis
software should allow for the use of the pooled internal standard to
facilitate the normalization and spot matching procedures. It is
usual for the software to incorporate some statistical tools to allow
for the assignment of spots of interest that can then be exported as
a pick list. These protein spots can then be excised for further anal-
ysis, such as by mass spectrometry to identify the protein.

3.5. Image Analysis

For spot picking, it is necessary to poststain a designated gel with
a total protein detection system (e.g., silver, Coomassie ® , or ide-
ally a fl uorescent stain such as Deep Purple or SYPRO Ruby) (see
Note 9).
The reason for this procedure is that if the original CyDye spot
coordinates were used, then there is the possibility that only the
protein with the dye attached will be picked (a small percentage of
the total protein) as this has an approximate 450-Da molecular
weight shift to a higher position in the gel (this is the same for all
three dyes). The bulk of the protein lies at a slightly lower molecular

3.6. Gel Processing
for Spot Picking