1. Sample preparation: Proteins are extracted from cells or tissues of interest. Protein concentrations are determined by protein assay and adjusted to the desired concentration.

2. Sample labeling with CyDye DIGE fluors: Equal amount of protein extract from paired samples were labeled by CyDye DIGE fluors (size and charge matched) respectively, and the spectrally resolvable dyes enable simultaneous co-separation and analysis of samples on a single multiplexed gel.

3. 2-D gel electrophoresis: Up to three samples can be simultaneously separated on a single 2-D gel, using isoelectric focusing (IEF) in the first dimension and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension.

4. Image acquisition: After electrophoresis, the gel is scanned using a Typhoon image scanner. Each scan reveals one of the CyDye signals (Cy2, Cy3 and Cy5).

5. Image analysis: ImageQuant software is used to generate the image presentation data including the single and overlay images. Then the images are subjected to DeCyder software analysis. DeCyder in-gel analysis software automatically locates and analyzes multiplexed samples in the same gel. It also allows complex analysis of multiple gels to provide comparative analysis and accurate measurement of differential protein expression.

6. Automated spot picking: After the spot picking design using DeCyder software, protein spots of interest can be automatically picked from the 2-D gel with the Ettan Spot Picker, followed by Protein Identification by Mass Spectrometry.


Please click for examples of 2-D DIGE image presentation and a sample report.