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Imaging and Analysis of DIGE Product Information Sheet, Rev B

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
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TAG: Imaging and Analysis DIGE Product Information Sheet Rev

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