Compare the Cy3 and Cy5 images with the Scatter Plot view (Analyze>Scatter Plot). Activate and inactivate normalization to view the effect of normalization.
The multi-channel viewer (View>Multi-Channel Viewer) gives an initial visualization of matched and unique spots in the three Cy-labeled images, but it is not necessary for the analysis. Overlay the desired images and select the Auto-Scale Image When Assigned checkbox to adjust the images to similar brightness and contrast.
The following color scheme is suggested: blue for the Cy2 image, green for the Cy3 image, and red for the Cy5 image.
Use Analysis Set Manager to create analysis sets that contain spots exhibiting significant protein regulation between the Cy3 and Cy5 samples (Analyze>Analysis Set Manager>Create). The Spot Review tool (Analyze>Spot Review) can be used to review these analysis sets and to verify that all the spots in each analysis set are defined as desired. A subset of these spots can be selected by clicking on their graphs and then selecting Create Analysis Set to establish the desired DIGE analysis set.
Carry out a separate spot detection on the SYPRO Ruby stained gel. Use the Automated Detection and Matching dialog box, and disable steps 3 and 4. It is not necessary for preparative gels to match perfectly to the corresponding DIGE gels in most cases the preparative and analytical gel images will appear very different. However, it should be possible to locate the proteins of interest that were targeted in the analytical gels in the