Ettan DIGE

Figure 3.2 Difference gel electrophoresis (DIGE). Ettan DIGE workflow three-color and two-color experiments including the internal standard. For fluorescence proteins tagging, two different CyDyes techniques are available. Minimal fluors allow consideration of three different CyDyes (Cy2, Cy3 and Cy5) in a multiplexing experiment.

CyDye Difference Gel Electrophoresis (DIGE) Fluors for Ettan DIGE (GE-Healthcare, Little Chalfont, UK) The dye tubes (Cy2, Cy3, Cy5) should be allowed to warm to room temperature for 5 min. Add 10 pL DMF (freshly opened) to each dye tube and mix. The tubes now contain 1 mM Cy2, Cy3, or Cy5 dye in DMF and should be vortexed vigorously for 30 s. Afterward, centrifuge the tubes for 30 s at 12,000 x g. The fluorochrome can now be used. Unused CyDye stock solutions should be returned to -20 °C as soon as possible and stored in the dark. 

Cy2, Cy3, and Cy5 can be detected using a Typhoon laser scanner (9200, 9210,9400, or 9410). The unfixed gels are scanned according to the Ettan Dige User manual (GE-Healthcare). 

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