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2D DIGE Protein Expression Profiling Procedure
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. 2D gel electrophoresis: Up to three samples can be simultaneously separated on a single 2D 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.
6. Quantitative analysis: comparative analysis of all spots using DeCyder ‘in-gel’ or ‘cross-gel’ analysis software. The protein expression ratios between different samples or different groups of samples will be generated.
7. Automated spot picking: After the spot picking design using DeCyder software, protein spots of interest can be automatically picked from the 2D gel with the Ettan Spot Picker, followed by Protein Identification by Mass Spectrometry.
8. Cluster Analysis: We can provide this for data sets of 50 or more proteins. Please see the following link for more information: http://www.appliedbiomics.com/Services/pathway.html
9. Validation: We can validate the 2D DIGE results with 2D Western Blot. Please see the following link for more information or call our tech support for additional details: http://www.appliedbiomics.com/Services/2d-western-blot.html
Please click for examples of 2D DIGE image presentation, sample report, and protein quantitation.