( Difference ...)Imaging and Analysis of DIGE Product Information Sheet, Rev B

Difference gel electrophoresis (DIGE) is a relatively straightforward application of differential labeling of protein samples using fluorescent dyes. The technique, originally published by Unlu et al. (1997), uses cyanine (Cy) dyes to differentially label proteins separated by 2-D gel electrophoresis. Following labeling and electrophoresis, excellent results can be achieved by scanning the multiplex gels with the Molecular Imager FX multiimager system and analyzing them with PDQuest 2-D analysis software.

The DIGE method is designed to compare two complex protein samples derived from two different growth conditions. One sample is labeled with Cy3 and the other with Cy5. A third dye, Cy2, is used to label a 50:50 mixture of the two samples, which becomes a standard for registering the images obtained with the other two dyes. Equal amounts of the three labeled protein mixtures are combined and run on the same 2-D polyacrylamide gel, eliminating any gel-to-gel variation.

Imaging
Image Acquisition With the Molecular Imager FX System
The Molecular Imager FX multiimager systems are well suited for image acquisition from gels containing Cy-dyed proteins. The laser and filter combinations provided ensure minimal cross-talk between the different fluors.

Lasers
The lasers required for the Cy dyes are the 488 nm external laser for Cy2, the 532 nm internal laser for Cy3, and the 635 nm external laser for Cy5.

Emission Filters
The required emission filters are 530BP30 for Cy2, 605DF50 for Cy3, and 695DF55 for Cy5.

Loading the Gels Into the Molecular 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 automated 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 matching 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 significant amount of saturated pixels, select Total Density in Gel Image instead.

Compare the Cy3 and Cy5 images with the Scatter Plot view (Analyze>Scatter Plot). Activate and inactivate normalization to view the effect of normalization.

Multi-Channel Viewer
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.

Analysis
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.

Preparative Gels
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 preparative gels. The proteins of interest will be those in the DIGE analysis sets. Since Cy-labeled and SYPRO Ruby stained gels are visibly different, they must be matched manually.

Matching Cy-Labeled Images With the SYPRO Ruby Stained Gel
Add the SYPRO Ruby image to the DIGE Matchset using the Matchset tool. Try matching the SYPRO image to the master image using the automated matching method first (Match>Automated Matching). If automated matching does not produce useful results, add some landmarks and rerun automated matching. Use Classic as the extended match method. If automated matching with added landmarks is unsuccessful, it will be necessary to carry out a manual match (Match>Classic Match Gel). Begin by manually landmarking the SYPRO Ruby image to the DIGE master image (Match> Landmark). Select as landmarks spots that are clearly in equivalent positions in the Cy2 and SYPRO Ruby gels whether or not they have the same intensities (Match>Landmark). Match the SYPRO Ruby image to the DIGE master image (Match>Classic Match Gel).

Locate Spots of Interest in the Preparative Gel
In order to map the spots of interest from the analytical gels to the preparative gel, verify that the spots are matched to the SYPRO Ruby stained gel using the Spot Review tool. Open the Spot Review tool (Analyze>Spot Review). Select the analysis set containing the interesting DIGE spots. Verify that these spots are matched to the preparative SYPRO Ruby stained gel. If the spots are not matched to the preparative gel, use manual matching (Match>Edit Matches>Manual Match) to match these spots.

Once the DIGE spots are matched to the SYPRO Ruby stained gel, proceed with isolation of the targeted Analysis Set spots using Bio-Rads ProteomeWorks Plus spot cutter. PDQuest software will direct automatic excision of the targeted proteins, track the excised spots through microtiter plate digestion and mass spectrometry protein identification, and allow automatic annotation of the gel image with the resulting protein identifications.

Reference
Unlu M et al., Difference gel electrophoresis: a single gel method for detecting changes in protein extracts, Electrophoresis 18, 20712077 (1997)

Cy is a trademark of Amersham Biosciences. SYPRO is a trademark of Molecular Probes, Inc.

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