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Imaging and Analysis of DIGE Product Information Sheet, Rev B

r Imager FX System
Since proteins are covalently labeled with the Cy dyes, post-run staining is not necessary for analysis. Bio-Rads Ready Gel, Criterion, and PROTEAN II precast gels must be removed from their cassettes and placed on the standard glass sample tray. Gels in glass cassettes may be scanned while in the cassettes as long as the glass has low fluorescence and the plates are not thicker than 11.5 mm. Removing at least the top glass plate prior to scanning is recommended. Scans obtained through top glass plates are at least 2-fold less intense than those obtained with the plates removed. Gels removed from their cassettes should be placed on the standard glass sample tray. For gels between thick glass plates, the multi-sample tray II should be used with an appropriate spacer to bring the sample into the proper focal plane for imaging.

Setting Up the Imaging Software
The Molecular Imager FX acquisition screen contains preset applications to automatically select the correct laser and filter combinations. Select Cy2, Cy3, and Cy5 from the preset applications located in the PDQuest acquisition window.

Automated Scanning
Once the applications are created, images can be acquired sequentially or automatically. Make sure that the acquisition frame is tightly wrapped around the gel image, and do not change the frame between successive scans. Since all three images are obtained from the same gel, it is essential that the gel not be moved between scans and that the acquisition frame be the same for all scans.

For sequential scanning, simply select the Cy3 setting in Channel 1 and scan. Repeat for Cy5 and Cy2. For automate
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