( atching procedures. Since al...)Imaging and Analysis of DIGE Product Information Sheet, Rev B

atching procedures. Since all of the images come from the same gel, matching is straightforward and well suited to the automatic functions of PDQuest. Matchsets created to compare the Cy3 and Cy5 images use the Cy2 image as the master image for the matching of spots across the images. Since the DIGE method is meant to be quantitative, intensity normalization between images is important to compensate for variations in spot intensity caused by systematic variations such as different characteristics of the lasers, filters, and dyes.

Transform the Images
Open all of the images pertinent to the experiment and transform as desired for visualization (Image>Transform).

Crop the Images
Trim away extraneous areas of the gel images using the Advanced Crop function (Image> Advanced Crop). Cropping will be efficient as long as the gel was not moved during image acquisition. It is recommended that you apply the Advanced Crop tool without the use of the placement crosshair. Accurate cropping is most important before using the multi-channel viewer to assess the similarities and differences among the three images.

Spot Detection and Matching
Automated detection and matching (Spots> Automated Detection and Matching) can be used for DIGE analysis. Assign the Cy2 image as the master and proceed with the Spot Detection Wizard to detect spots on all the gel images.

Normalization
Normalization of image data is important for meaningful quantitative comparisons of the different images. With the Matchset open, enable normalization from the Analyze menu (Analyze>Normalize) and select Total Quantity in Valid Spots. If any of the images contain a significa
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