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2D DIGE Protein Array And Mass Spectrometry

 

 

 

Our 2D DIGE Protein Array / Mass Spectrometry proteomics services offer a powerful platform in studying the high complexity proteome of all biological systems. While 2D gel continues to be one of the most versatile and widely used techniques, a modified version, 2D-DIGE, which labels different protein samples with up to three fluorescent tags, offers greater sensitivity and reproducibility over conventional 2D-PAGE gels.

Main applications:

- Identifying therapeutic or diagnostic markers in different diseases
- Identifying key regulators in major biological pathways
- Studying post-translational modifications such as phosphorylation, glycosylation etc
- Determining HCP antibody coverage and detecting HCP contaminants

Our 2D-DIGE Protein Array offers significant ADVANTAGES over the standard 2D gel and other protein array platforms:

- High sensitivity: Fluorescent dyes have a sensitivity of 0.2 ng/spot, versus the sensitivity of coomassie at 100 ng/spot or silver staining at 1 ng/spot
- Broad spectrum: Each gel can resolve ~5000 protein spots allowing a wide dynamic range detection/quantitation of low abundant proteins, large proteins and small peptides.
- High accuracy: The extremely high spot resolution enables accurate spot quantitation. Differences in protein expression as small as 10% can be detected
- Fewer number of gels: Each gel can accommodate up to 3 different samples
- Cost effective: The 2D DIGE price covers from sample preparation to publication-ready data and complimentary consultation with your project leader
- Fast turn-around: 1 week

 

2D DIGE Protein Array / Mass Spectrometry Procedure


2D DIGE Protein Expression Work Flow

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 fluorescent dyes: Equal amount of protein extract was labeled by fluorescent dyes (size and charge matched), and the spectrally resolvable dyes enable simultaneous co-separation and analysis of up to 3 samples on a single multiplexed gel.

3. 2D gel electrophoresis: Up to 3 samples can be simultaneously separated on a single 2D gel, with isoelectric focusing (IEF) in the 1st dimension and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in the 2nd 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: Following 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: A complimentary service for projects with 50 or more identified proteins.

9. Validation: We offer 2D Western Blot to validate the 2D DIGE data.

Please view an example of sample report.

If you are interested, please follow these steps to obtain more information and order this service.