( d scanning set three of the ...)Imaging and Analysis of DIGE Product Information Sheet, Rev B

d scanning, set three of the channel tabs to conform to the different Cy dyes (e.g., set Channel 1 for Cy3, Channel 2 for Cy5, and Channel 3 for Cy2). The order does not matter. The Enable Channel box must be selected in each channel to automate the acquisition. A green check mark appears for each enabled channel.

Optimizing Imaging
To save time by avoiding scanning areas outside the gel, a quick initial scan at 800 m resolution should be done to find the location of the gel and the approximate intensity of the spots. Select the Highlight Saturated Pixels box to aid in interpreting sample intensity. No more than a few of the brightest spots should contain saturated pixels. If an image contains too many saturated pixels, reduce the photomultiplier tube (PMT) voltage. Change the PMT voltage through the Select Application pop-up window by selecting either a different preset sample intensity setting (e.g., high, medium, or low) or a custom voltage for more refined control. Keep in mind that high sample intensity (saturated pixels) requires a lower PMT voltage, and a low sample intensity requires a higher PMT voltage.

Preparative Gels and Protein Identification
For optimal mass spectrometry results, it is recommended that you use SYPRO Ruby stained preparative gels to obtain enough protein for identification.

Load the SYPRO Ruby stained gel into the Molecular Imager FX as described earlier. In the Molecular Imager FX acquisition window, select the SYPRO Ruby preset application and scan as before for the Cy-dyed gels.

Analysis
Image Analysis With PDQuest Software
PDQuest analyzes DIGE experiments with standard gel m
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