Evaluating the Efficacy of Subcellular Fractionation of Blast Cells Using Live Cell Labeling and 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 330

3.5. Image Acquisition
and Analysis

1. Stop the SDS PAGE when the dye front reaches the bottom

of the gel (approximately 16 h). Open the lid and begin disas-
sembling the tank by removing a blank cassette from the rear
or the apparatus. Place the cassettes on a gel rack and rinse the
cassettes thoroughly with deionized water. Lay the gel cassette
on the bench and open the cassette carefully. The gel should
still attach to the glass plate. Hold the edges of the plastic back-
ing and start pulling the gel away from the glass plate. Put the
gel onto the plastic coversheet that comes with the gel. This
helps transporting the gel and reducing exposure to contami-
nants (such as keratin).

2. Scan the gels using the Typhoon Trio scanner. Clean the glass

scanner surface with ethanol and water. Squeeze a small vol-
ume of water on the glass surface. Remove the gel coversheet,
hold the edges of the gel plastic backing, and slowly lay the gel
downward toward the scanner glass surface without introduc-
ing bubbles between the glass surface and the gel. Place a clean
low-fl uorescent glass plate on top of the gel to hold the gel fl at.
Scan using the settings outlined in Table 3 .

3. The images can be qualitatively visualized using ImageQuant

TL. The brightness and contrast settings for each gel require
adjustment to obtain an optimal picture. By examining the
relative Cy3 (LCL) and Cy5 (post-fractionation labeling) pat-
terns for the membrane and cytosolic fractions, the effi cacy of
subcellular fractionation can be assessed. As shown in Fig. 2 ,
the majority of the Cy3-labeled proteins were enriched in the
membrane fraction with very limited Cy3-labeling of proteins
isolated in the cytosolic fraction. Furthermore, the similarities
observed between the Cy5- and Cy3-labeled components of
the membrane fraction indicated minimal cytosolic contamina-
tion. Potential improvements to this experimental design that
may allow for the quantifi cation of differences between live-
cell-labeled membrane and cytosolic proteins are outlined
below (see Note 1 and Fig. 3 ).

Table 3
Scanner setting for the Typhoon Trio

CyDye Excitation (nm) Emission (nm) Resolution ( m m) PMT (V)

Cy3 532 (green) 580 100 700

Cy5 633 (red) 670 100 450